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US20250263654A1 - Primordial germ cells - Google Patents

Primordial germ cells

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US20250263654A1
US20250263654A1 US18/569,550 US202218569550A US2025263654A1 US 20250263654 A1 US20250263654 A1 US 20250263654A1 US 202218569550 A US202218569550 A US 202218569550A US 2025263654 A1 US2025263654 A1 US 2025263654A1
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cells
pluripotent stem
stem cells
cell
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Todd C. McDevitt
Ivana Vasic
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J David Gladstone Institutes
University of California San Diego UCSD
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J David Gladstone Institutes
University of California San Diego UCSD
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Assigned to THE J. DAVID GLADSTONE INSTITUTES, A TESTAMENTARY TRUST ESTABLISHED UNDER THE WILL OF J. DAVID GLADSTONE reassignment THE J. DAVID GLADSTONE INSTITUTES, A TESTAMENTARY TRUST ESTABLISHED UNDER THE WILL OF J. DAVID GLADSTONE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MCDEVITT, TODD C.
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Definitions

  • the pluripotent stem cells employed can be human induced pluripotent stem cells (hiPSCs)).
  • the PSCs can be genetically modified (e.g., to repair genetic mutations or to facilitate PGC differentiation).
  • the PSCs can be genetically modified to express genes involved in PGC specification or genetically modified to make the PSCs more susceptible to PGC differentiation.
  • such genetic modification is not needed to produce primordial germ cells from PSCs.
  • an effective method is described herein that involves basolateral stimulation of human induced pluripotent stem cells with BMP.
  • the methods can involve seeding PSCs into vessels that provide BMP with basolateral access to the PSCs.
  • pluripotent stem cells can be supported on a porous surface in a culture medium that contains BMP. This method does not require genetic modification of the pluripotent stem cells to provide primordial germ cells.
  • the porous surface can be a membrane that freely allows nutrients and morphogens (e.g., proteins such as BMP) to circulate through the membrane.
  • morphogens e.g., proteins such as BMP
  • One type of culture apparatus that includes a porous surface for culture of the cells is a transwell culture system. Examples of materials that can be used for the porous surface include porous polycarbonate, polyester (PET), and/or collagen-coated polytetrafluoroethylene (PTFE) materials.
  • the pluripotent stem cells can be induced pluripotent stem cells (iPSCs), such as human induced pluripotent stem cells (hiPSCs).
  • iPSCs induced pluripotent stem cells
  • hiPSCs human induced pluripotent stem cells
  • Cells can be obtained from a selected subject, iPSCs can be generated from the subject's cells, and those iPSCs can then be converted into primordial germ cells. Mature germ cells can be generated from the primordial germ cells and used for in vitro fertilization to provide an embryo that can be implanted for gestation in a female.
  • the pluripotent stem cells or the induced pluripotent stem cells can be autologous or allogenic to a subject who desires in vitro fertilization.
  • the subject can be any mammalian or avian subject.
  • the methods and systems can be used to provide primordial germ cells for domesticated animals, wild animal species, endangered animal species (e.g., an animal on an endangered species list), as well as
  • the pluripotent stem cells can be genetically modified.
  • the pluripotent stem cells can be genetically modified to correct a genetic defect.
  • the pluripotent stem cells can be genetically modified to reduce the expression or function of an endogenous tight junction gene.
  • a tight junction gene can be at least one endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • At least one tight junction allele of any of these genes can be genetically modified.
  • two tight junction alleles of any of these genes can be genetically modified.
  • the pluripotent stem cells can be induced pluripotent stem cells (iPSCs), such as human induced pluripotent stem cells (hiPSCs).
  • iPSCs induced pluripotent stem cells
  • hiPSCs human induced pluripotent stem cells
  • the pluripotent stem cells can be genetically modified.
  • the pluripotent stem cells can be genetically modified to correct a genetic defect.
  • the methods and systems described herein can involve culturing cells on porous surfaces (e.g., a transwell) under conditions that provide growth of the cells.
  • a porous surface e.g., transwell
  • Such a porous surface can have an apical compartment as well as a basolateral compartment.
  • the pluripotent stem cells can be one the porous surface in the apical compartment and receive BMP from at least a basolateral compartment.
  • FIG. 1 D illustrates fluorescent measurements of media aliquots taken over time from the basolateral side of a transwell in which a wild type cell layer or a ZO1 knockdown cell layer was maintained after addition of FITC-dextran to the apical side of the transwell.
  • the wild type cell layer forms a membrane that is less permeable to the FITC-dextran than is the ZO1 knockdown cell layer.
  • This graph illustrates how barrier function and ability to preclude diffusion of molecules from one side of a cellular monolayer to the other (apical to basolateral diffusion) is disrupted by ZO1 knockdown.
  • FIG. 1 E graphically illustrates transepithelial resistance in wild type and ZO1-knockdown cells treated for 5 days with Doxycycline (2 uM), indicating loss of barrier function with ZO1 knockdown.
  • FIG. 1 F shows images of wild type and ZO1 knockdown cells immunostained for the nuclear marker Lamin-B1 (LMNB1) or for cytovillin (EZRIN), an apical polarity protein. As shown, expression of EZRIN is attenuated with ZO1 knockdown cells, indicating loss of apical/basolateral polarity.
  • FIG. 1 G shows chromosomal images illustrating the karyotype of a ZO1 WTC-LMNB1-GFP-CRISPRi (male ZO1 knockdown line).
  • FIG. 1 H illustrates karyotyping analysis of expression from chromosomal loci demonstrating that all genetically modified lines used to validate results in this study are karyotypically normal, including the ZO1 WTC-LMNB1-GFP-CRISPRi (male ZO1 knockdown line), ZO1 WTB-CRISPRi-Gen1B (female ZO1 knockdown line) and ZO1 WTC-NANOS3-mCHERRY (male ZO1 knockdown line, with PGC reporter).
  • ZO1 WTC-LMNB1-GFP-CRISPRi male ZO1 knockdown line
  • ZO1 WTB-CRISPRi-Gen1B female ZO1 knockdown line
  • ZO1 WTC-NANOS3-mCHERRY male ZO1 knockdown line, with PGC reporter
  • FIG. 2 A- 2 E illustrate the method by which PGCLCs (primordial germ like cells, designated “like” because they are generated in vitro) are generated from ZO1 wild type and ZO1 knockdown hiPSCs.
  • FIG. 2 A illustrates that as a result of impaired barrier function, ZO1 knockdown hiPSCs lose polarized response to BMP4, enabling activation of pSMAD1 when BMP4 is presented apically (apical presentation is typical in standard/non-transwell culture).
  • FIG. 2 B is a schematic illustrating methods for determining specification bias, which was used to assay the ZO1 knockdown cells in comparison to ZO1 wild type cells.
  • FIG. 2 C shows the results of the specification bias assay delineated in FIG.
  • FIG. 2 E shows replicate immunofluorescent staining of control ( ⁇ DOX) and ZO1 knockdown (+DOX) cells after treatment with BMP4 for 48 hours to detect a panel of PGC markers (BLIMP1, SOX17, and TFAP2C). Double positive staining was used to identify primordial germ cell like cells (PGCLCs; which are primordial germ cells generated in vitro). SOX2 is not a PGC marker and was shown as a negative control. In the original SOX2 was stained blue, TFAP2C was stained blue, BLIMP1 was stained red, and SOX17 was stained green.
  • PGCLCs primordial germ cell like cells
  • FIG. 3 schematically illustrates that PGC (also called PGCLC) differentiation can be achieved via ZO1 silencing, pharmacological inhibition of ZO1, or by growth of cells on transwell membranes in the presence of BMP4.
  • PGC also called PGCLC
  • Such growth of cells on transwell membranes requires no chemical and no structural perturbation cells, and instead is mediated by basolateral stimulation by BMP.
  • BMP-SMAD1 pathway Illustrated in FIG. 2 A .
  • FIG. 2 A a typical epithelial cell layer in culture is schematically illustrated on the left, which forms tight junctions maintained by ZO1 and which does not produce PGCs (PGCLCs) upon stimulation with BMP4.
  • FIG. 4 schematically illustrates the role of Zonula occludens-1 (ZO1, also called TJP1) within cells and how ZO1 maintains epithelial structure.
  • ZO1 is a tight junction protein expressed in primed pluripotent stem cells in standard in vitro culture.
  • ZO1 forms dual-purpose adhesion plaques that endow an epithelium with both barrier and partitioning functions (polarity/directionality), thereby attenuating responses to morphogen signals (such as BMP4).
  • FIG. 5 A- 5 H illustrate that unconfined human iPSC colonies undergo radial gastrulation-like patterning with loss of ZO1 on the colony edge.
  • FIG. 5 A illustrates a method where hiPSCs were aggregated into pyramidal wells, subsequently plated, and induced with BMP4 for 48 hours.
  • FIG. 5 B illustrates that unconfined colonies of wild type hiPSCs undergo radial patterning of gastrulation-associated markers after 48 hrs of BMP4 stimulation.
  • FIG. 5 C shows immunofluorescence images of a wild type colony edge, showing loss of ZO1 and gain of pSMAD1 at the colony edge.
  • FIG. 5 E shows images of unconfined and low/high density micropatterned colonies, with a comparison of ZO1 and pSMAD1 expression in these wild type colonies.
  • FIG. 5 F shows images of unconfined wild type colonies illustrating that they maintain honeycomb ZO1 expression over time.
  • FIG. 5 G graphically illustrates cell density measurements in unconfined wild type colonies, with a projected density curve for micropatterned colonies (assuming density of 5,000 cells/mm 2 upon induction with BMP4). Epithelial range, based on structure of cell-cell junction pattern, was estimated to be in the range of 3,000-10,000 cells/mm 2 .
  • FIG. 5 H shows images of wild type cellular monolayers illustrating ZO1 and pSMAD1 expression as a function of cell density in monolayer culture. The epithelial structure (honeycomb cell-cell junction pattern) is lost and pSMAD1 activation is increased as cell density increases.
  • FIG. 6 A- 6 I illustrate that ZO1 knockdown (ZKD) causes ubiquitous and sustained phosphorylation of SMAD1 throughout cellular colonies over time.
  • FIG. 6 A is a schematic illustrating that CRISPRi knockdown of ZO1 increases signaling protein accessibility.
  • FIG. 6 B shows a Western blot illustrating ZO1 protein loss in the ZO1 knockdown cell lines. The WTB (female) and WTC (male) cells are parental hiPSC lines.
  • FIG. 6 C shows immunofluorescence images and brightfield images illustrating morphological differences between ZO1 wild type and ZO1 knockdown cells.
  • FIG. 6 D graphically illustrates changes in nuclear height, area, cell density, and growth rate of ZO1 wild type and ZO1 knockdown cells.
  • FIG. 6 A- 6 I illustrate that ZO1 knockdown (ZKD) causes ubiquitous and sustained phosphorylation of SMAD1 throughout cellular colonies over time.
  • FIG. 6 A is a schematic illustrating that CRISPRi knockdown of
  • FIG. 6 E graphically illustrates the fraction of pSMAD1+ cells over time, normalized to expression of LMNB1 (n ⁇ 3), in populations of ZO1 wild type and ZO1 knockdown cells.
  • FIG. 6 F shows immunofluorescence images illustrating maintained and ubiquitous phosphorylation of SMAD1 in ZO1 knockdown (ZKD) cells compared to ZO1 wild type cells over the course of 48 hours.
  • FIG. 6 G is a schematic illustrating a FITC-dextran diffusion assay.
  • ZWT ZWT
  • ZKD ZKD
  • FIG. 6 H graphically illustrates the fluorescence observed from the basolateral compartment over time using the method illustrated in FIG. 6 G .
  • FIG. 6 I graphically illustrates transepithelial electrical resistance (TEER) measurements in ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) monolayers.
  • TEER transepithelial electrical resistance
  • FIG. 7 A- 7 N illustrate that ZO1 knockdown (ZKD) cells are biased toward differentiation into PGCs.
  • FIG. 7 A is a schematic showing the inventors' predictions regarding spatial emergence of distinct lineages arising in ZO1 wild type (ZWT; top) and ZO1 knockdown (ZKD; bottom) colonies exposed to BMP4 under a reaction diffusion (RD)/positional information (PI) patterning model.
  • FIG. 7 B shows immunofluorescence images of canonical germ lineage markers LMNB1, CDX2, SOX2, TBXT in ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells after 48 hours of stimulation with BMP4.
  • FIG. 7 C graphically illustrates the fraction of cells positive for expression of the markers shown in FIG.
  • FIG. 7 B in wild type (ZWT) and ZO1 knockdown (ZKD) cells.
  • FIG. 7 D shows a volcano plot of RNA sequencing data illustrating log fold changes of SOX2, TBXT, and CDX2.
  • FIG. 7 E graphically illustrates RNA sequencing data illustrating expression levels of canonical germ layer markers in ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells after 48 hours of stimulation with BMP4.
  • FIG. 7 F illustrates unbiased clustering of the top 16 differentially expressed genes between ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells, highlighting increases in PGC-related genes.
  • FIG. 7 G shows immunofluorescent images of LMNB1, and PGC markers BLIMP1, SOX17, TFAP2C in ZO1 wild type (ZWT) and ZO1 knockdown cells (ZKD) after 48 hours of stimulation with BMP4.
  • FIG. 7 H illustrates that pSMAD1 expression is only activated upon basolateral (top row) BMP4 stimulation in wild type ZO1 cells, but not by apical BMP4 stimulation. However, both apical and basolateral stimulation by BMP activates pSMAD1 in ZO1 knockdown (ZKD) cells.
  • FIG. 7 I graphically illustrates levels of BMP receptor expression in ZO1 wild type and ZO1 knockdown cells as observed from RNA sequencing data.
  • FIG. 7 J graphically illustrates the fold change in secreted morphogens at 12 hours of BMP4 stimulation, showing significant increases in Noggin (NOG) in the ZO1 knockdown (ZKD) cells that are not seen in ZO1 wild type cells, as detected by qPCR.
  • FIG. 7 K shows images of cells illustrating the positioning of the Golgi in ZO1 wild type (left) and ZO1 knockdown (right) cells. Z-stacks revealed that in both cell types, the Golgi sits on top of the nucleus on the apical side of the cell, indicating that polarity of the ZO1 knockdown cells is still intact.
  • FIG. 7 J graphically illustrates the fold change in secreted morphogens at 12 hours of BMP4 stimulation, showing significant increases in Noggin (NOG) in the ZO1 knockdown (ZKD) cells that are not seen in ZO1 wild type cells, as detected by qPCR.
  • FIG. 7 K shows images of cells illustrating the positioning
  • FIG. 7 L graphically illustrates the fluorescence intensity of immunostained Golgi as a function of the distance from the nuclear center of ZO1 wild type and ZO1 knockdown cells, indicating that the Golgi sits on top of the nucleus on the apical side of both cell types.
  • FIG. 7 M shows images of immunofluorescent-stained ZO1 wild type cells (left) and ZO1 knockdown cells (right), illustrating that ZO1 knockdown cells lost apical Ezrin expression (dark area delineated by a white dashed line). Even in regions where Ezrin is present, the Ezrin overlaps significantly with BMPR1A (a basolateral BMP receptor).
  • BMPR1A a basolateral BMP receptor
  • FIG. 8 A- 8 H illustrate ZO1 knockdown cells have a bias for PGC differentiation.
  • FIG. 8 A shows images of immunofluorescent-stained ZO1 wild type and ZO1 knockdown cells illustrating expression of LMNB1, BLIMP1, SOX17, TFAP2C after 48 hours and 72 hours of stimulation with BMP4.
  • FIG. 8 B graphically illustrates the percent of ZO1 wild type and ZO1 knockdown cellular nuclei that exhibit expression of the indicated PGC markers (n ⁇ 3).
  • FIG. 8 C illustrates expression of canonical pluripotency markers in ZO1 wild type and ZO1 knockdown cells prior to BMP4 stimulation.
  • FIG. 8 A shows images of immunofluorescent-stained ZO1 wild type and ZO1 knockdown cells illustrating expression of LMNB1, BLIMP1, SOX17, TFAP2C after 48 hours and 72 hours of stimulation with BMP4.
  • FIG. 8 B graphically illustrates the percent of ZO1 wild type and ZO1 knockdown cellular nucle
  • FIG. 8 D illustrates methylation levels of ZO1 wild type versus ZO1 knockdown cells; the data were from whole genome bisulfite sequencing data.
  • FIG. 8 E shows images of immunofluorescent-stained cells illustrating expression of LMNB1, BLIMP1, SOX17, TFAP2C in ZO1 wild type and ZO1 knockdown cells after 48 hours and 72 hours of stimulation with BMP4 in a female hiPSC line.
  • FIG. 8 F graphically illustrates the percent of ZO1 wild type and ZO1 knockdown cellular nuclei that exhibit expression of PGC markers (n ⁇ 3) in a female hiPSC line.
  • FIG. 8 G illustrates unbiased clustering of top 16 differentially expressed genes between ZO1 wild type and ZO1 knockdown cells in the pluripotent condition.
  • FIG. 8 H illustrates probe methylation levels between ZO1 wild type and ZO1 knockdown cells gathered from whole genome bisulfite sequencing data, probes with significant differences in methylation are darkly shaded.
  • FIG. 9 A- 9 B illustrate that ZO1 knockdown-related PGCLC bias is a product of signaling, not changes in pluripotency.
  • FIG. 9 A shows images of immunofluorescent-stained ZO1 wild type (top) and ZO1 knockdown (bottom) cells illustrating pSMAD1 expression after basolateral BMP4 stimulation for timepoints between 0-48 hrs when the cells were grown on the transwell membranes.
  • FIG. 9 A- 9 B illustrate that ZO1 knockdown-related PGCLC bias is a product of signaling, not changes in pluripotency.
  • FIG. 9 A shows images of immunofluorescent-stained ZO1 wild type (top) and ZO1 knockdown (bottom) cells illustrating pSMAD1 expression after basolateral BMP4 stimulation for timepoints between 0-48 hrs when the cells were grown on the transwell membranes.
  • FIG. 9 A shows images of immunofluorescent-stained ZO1 wild type (top) and ZO1 knockdown (bottom) cells illustrating
  • 9 B shows images of immunofluorescent-stained ZO1 wild type and ZO1 knockdown cells illustrating expression of LMNB1, BLIMP1, SOX17, TFAP2C when the cells were grown on transwell membranes with 48 hrs of bi-directional (apical and basolateral) stimulation with BMP4 at concentrations between 5-50 ng/ml.
  • PSCs pluripotent stem cells
  • hiPSCs human induced pluripotent stem cells
  • the compositions and methods provide useful numbers of primordial germ cells (PGCs) with an efficiency of about 50-60% and without the need for three-dimensional (3D) suspension or bioreactor culturing procedures.
  • the epithelial barrier structure of the induced pluripotent stem cells is modified by the methods described herein either during differentiation by basolateral exposure to BMP, by exposure to tight junction inhibitors, or by using CRISPR interference (CRIPSRi) to inhibit, knock down, or knockout one or more tight junction genes or tight junction proteins.
  • CRISPR interference CRISPR interference
  • PSCLCs primordial germ cells
  • reprogramming methods involve manipulating primed PSCs to a more na ⁇ ve PSC state that structurally/transcriptionally/epigenetically resembles the apolar inner cell mass/pre-implantation epiblast (E5-E9).
  • This has been done through transient delivery of transgenes via expression vectors or by introducing RNA, or through exposure of the primed PSCs to various cytokines/histone deacetylases, and other chemicals and/or biological molecules (e.g., LIF, SCF, EGF, Activin A, CHIR99021).
  • pluripotent stem cells e.g., human induced pluripotent stem cells (hiPSCs)
  • pluripotent stem cells e.g., human induced pluripotent stem cells (hiPSCs)
  • hiPSCs human induced pluripotent stem cells
  • pluripotent stem cells can be cultured under conditions that transiently inhibit relevant tight junction proteins, for example, by knockdown of tight junction protein expression or through pharmacological inhibition of tight junction protein functions.
  • tight junctions are assembled via the protein ZO1.
  • Such tight junctions are used by cells to split the cell into two “sides”: the apical side and the basolateral side.
  • Apical refers to the outward-facing side(s) of a cell, which have more tight junctions than the basolateral side of cell.
  • Basolateral refers to the inward-facing side(s) of a cell.
  • Tight junctions can prevent diffusion of proteins and other small molecules between these two domains, thereby acting as a barrier.
  • Most morphogen receptors are basolateral (facing away from the media).
  • cells when cells are cultured so that at least one side rests or attaches to a surface, those cells are rendered partially or completely inaccessible to signals present in the media.
  • individual free floating cells may survive briefly in suspension, they do not survive for long.
  • Cells can be cultured for a while as aggregates in suspension but the same problems exist for aggregated cells as for cells maintained on solid surfaces-tight junctions are present on the apical sides of aggregated cells. Even when aggregated cells are disassociated, the tight junctions will quickly reassemble upon reaggregation of the cells.
  • Aggregated cells therefore have the same barrier/receptor access problems as cells cultured on solid surfaces-morphogens in the media are not taken up, or only occasionally take up, because the tight junctions on the apical surfaces block such uptake.
  • cellular differentiation is heterogeneous because stochastic signal pathway activation occurs.
  • Reducing the inhibiting tight junction formation or bypassing tight junctions or as described herein, for example by ZO1 knockdown or by basolateral stimulation (e.g., by growing cells on a transwell), provides homogeneous and sustained signal pathway activation.
  • Such reduction/removal of tight junctions is useful because signal pathway activation in the cells can specifically be controlled.
  • the culture methods described herein therefore optimize the PGC differentiation, providing the least expensive and fastest differentiation protocol to generate PGCs.
  • primordial germ cells can be generated from human induced pluripotent stem cells (e.g., hiPSCs) by incubating the PSCs in vessels that allow BMP to basolaterally contact the PSCs.
  • pluripotent stem cells can be used, including induced pluripotent stem cells (iPSCs), embryonic stem cells, embryonic stem cells made by somatic cell nuclear transfer (ntES cells), or embryonic stem cells from unfertilized eggs (parthenogenesis embryonic stem cells, pES cells).
  • the apical cell surface refers to the surface of a monolayer of cells that faces the culture medium.
  • the apical surface does not include the cell surface that contacts the culture plate or the culture vessel or that contacts an aggregated cell mass.
  • the basolateral cell surface refers to everything below the apical surface that can freely contact cell media. Hence the basolateral cell surface does not include the sides or the surfaces upon which the cells rest or that contact a solid surface or an aggregated cell mass. When cells are grown/maintained in a monolayer, the basolateral surface does not include the base of the cells that rest on a solid surface, or where the cells are laterally in contact with each other.
  • the cell base and the cell apical surfaces are generally on opposite sides of the cells.
  • the base of the PSCs can rest upon a porous surface.
  • the porous surface supports the cells.
  • the porous surface can have pores of any pore size so long as the cells cannot pass through the pores.
  • An example of a pore size range that can be used is about 0.4 ⁇ m to about 8.0 ⁇ m.
  • Such a porous surface can be a membrane.
  • culture medium containing BMP can be placed in a vessel or in wells of a culture plate.
  • a membrane e.g., transwell insert
  • the PSCs can be seeded onto the membrane (e.g., of a transwell plate compartment).
  • the cell medium below the cells (the basolateral compartment) therefore contains BMP.
  • the membrane can be conditioned prior to use.
  • the membrane can be incubated with extracellular matrix protein (e.g., Matrigel), and the extracellular matrix protein can be removed (e.g., by aspiration) from the membrane prior to seeding the PSCs onto the membrane.
  • extracellular matrix protein e.g., Matrigel
  • the PSCs can be seeded at various densities.
  • the PSCs can be seeded at cell densities of about 10 cells/mm 2 to 10,000 cells/mm 2 , or about 100 cells/mm 2 to 9,000 cells/mm 2 , or about 200 cells/mm 2 to 8,000 cells/mm 2 , or about 400 cells/mm 2 to 6,000 cells/mm 2 , or about 500 cells/mm 2 to 5,000 cells/mm 2 .
  • the PSCs can be seeded at cell densities of at least about 100 cells/mm 2 , or at least about 300 cells/mm 2 , or at least about 700 cells/mm 2 .
  • Primed pluripotent cell culture medias can be used. Examples include mTESR, MEF conditioned media, StemFit, StemPro, or E8.
  • the culture media used in the apical compartment need not contain BMP. However, the culture media used in the basolateral compartment does contain BMP2, BMP4, or a combination thereof. Depending on pore size of the transwell membranes used, BMP4 can sometimes diffuse to the apical compartment, however this does not affect PGCLC differentiation.
  • the BMP can be used in the basolateral culture media in various amounts.
  • BMP can be included in the basolateral culture media in amounts of at least 0.1 ng/ml, or at least 1 ng/ml, or at about 2 ng/ml or at least 5 ng/ml, or at least 10 ng/ml, or at least 20 ng/ml, or at least 25 ng/ml, or at least 30 ng/ml, or at least 35 ng/ml, or at least 40 ng/ml, or at least 50 ng/ml.
  • the BMP is used in the culture media in amounts less than 200 ng/ml, or less than 150 ng/ml, or less than 100 ng/ml, or less than 75 ng/ml, or less than 60 ng/ml.
  • BMP BMP in contact with the basolateral sides of cells modifies epithelial structures those cells to thereby facilitate their differentiation into primordial germ cells.
  • the starting cells can be induced to form pluripotent stem cells using either genetic or chemical induction methods.
  • methods for generating human induced pluripotent stem cells include those described by U.S. Pat. No. 8,058,065 (Yamanaka et al.), WO/2019/165988 by Pei et al., and U.S. Patent Application No. 20190282624 by Deng et al.
  • Induced PSC can also be generated through chemical reprogramming, via JNK pathway inhibition as illustrated by Guan et al. (Nature 605:325-331 (2022)).
  • a ROCK inhibitor can be used in the iPSC culture medium, especially prior to incubation with BMP.
  • the ROCK inhibitor can be Y-27632, which is a cell-permeable, highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK).
  • ROCK protein kinase
  • Y-27632 can improve survival of stem cells when they are dissociated to single cells and after thawing the stem cells. Y-27632 can also reduce or block apoptosis of stem cells.
  • the ROCK inhibitor can be used in the culture media in amounts of at least 0.5 uM, or at least 1.0 uM, or at least 2.0 uM, or at least 3.0 uM, or at least 4.0 uM, or at least 5.0 uM, or at least 6.0 uM, or at least 7.0 uM, or at least 8.0 uM, or at least 9.0 uM, or at about 10 uM.
  • the ROCK inhibitor is used in the culture media in amounts less than 30 uM, or less than 25 uM, or less than 20 uM, or less than 15 uM.
  • the ROCK inhibitor can be used in the culture media when the hiPSCs are initially seeded into the vessel (e.g., wells) where the primordial germ cells will be generated. However, the ROCK inhibitor can be removed when the culture media is replaced with media containing BMP.
  • Epithelial structures are maintained by tight junctions, via key tight junction scaffolding proteins, such as the Zonula-occludens (ZO) family of proteins. Tight junctions form dual-purpose adhesion plaques that endow an epithelium with both barrier and partitioning functions (polarity/directionality) (see FIG. 4 ). Disruption of epithelial tissue structure and apical/basolateral polarity specifically, as illustrated herein, is a key method for generating primordial germ cells.
  • tight junction scaffolding proteins such as the Zonula-occludens (ZO) family of proteins.
  • ZO Zonula-occludens
  • tight junction proteins in the PSCs can be inhibited or modified (knocked down or knocked out) to facilitate generation of primordial germ cells.
  • the PSCs or incipient mesoderm-like cells iMeLCs
  • the cells can be cultured with BMP.
  • experiments described herein show that treatment of adherent cultures of ZO1/TIP1 knockdown cells with BMP-4 for 48 hours yielded high numbers of PGC like-cells (PGCLCs).
  • tight junction inhibitors examples include PTPN1 (Tyrosine-protein phosphatase non-receptor type 1), acetylaldehyde, genistein, protein phosphatase 2 (PP2), Clostridium perfringens enterotoxins (and their derived mutants), monoclonal antibodies against Claudin-1 (75A, OM-7D3-B3, 3A2, 6F6), monoclonal antibodies against Claudin-6 (IMAB027), Claudin-2 (1A2), monoclonal antibodies against Claudin-5 (R9, R2, 2B12), monoclonal antibodies against Occludin (1-3, 67-2), and combinations thereof.
  • PTPN1 Tyrosine-protein phosphatase non-receptor type 1
  • PP2 protein phosphatase 2
  • Clostridium perfringens enterotoxins and their derived mutants
  • monoclonal antibodies against Claudin-1 75A, OM-7D3-B3, 3A2, 6F
  • Chelators can also be used as tight junction inhibitors, including calcium chelators.
  • one or more of the following chelators can be used: chelator is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), dimercaptosuccinic acid, dimercaprol, or a combination thereof.
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol-bis( ⁇ -aminoethyl ether)-N,N,N′,N′-tetraacetic acid
  • dimercaptosuccinic acid dimercaprol, or a combination thereof.
  • tight junction proteins can be knocked down or knocked out before BMP treatment to facilitate generation of primordial germ cells.
  • tight junction genes or tight junction proteins to be modified, inhibited, knocked down or knocked out include zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, CLDN7.
  • Pluripotent stem cells primarily express ZO1.
  • the TIPI gene is on chromosome 15 (location 15q13.1; NC_000015.10 (29699367 . . . 29969049, complement).
  • a nucleotide sequence that encodes the ZO1 polypeptide with SEQ ID NO: 1 is available as European Nucleotide Archive accession no. L14837, provided below as SEQ ID NO:2.
  • Z (2) polypeptide An amino acid sequence for a human zonula occludens-2 (Z (2) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. Q9UDY2.2; UNIPROT accession no. Q9UDY2) and shown below as SEQ ID NO:3.
  • the TJP2 gene is on chromosome 9 (location NC_000009.12 (69121006 . . . 69255208)).
  • a nucleotide sequence that encodes the ZO2 polypeptide with SEQ ID NO:3 is available as European Nucleotide Archive accession no. L27476, provided below as SEQ ID NO: 4.
  • NCBI accession no. EAW69293.1; UNIPROT accession no. 095049 An amino acid sequence for a human zonula occludens-3 (ZO3) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. EAW69293.1; UNIPROT accession no. 095049) and shown below as SEQ ID NO:5.
  • the TJP3 gene is on chromosome 19 (location NC_000019.10 (3708384 . . . 3750813)).
  • a nucleotide sequence that encodes the ZO3 polypeptide with SEQ ID NO:5 is available as European Nucleotide Archive accession no. AK091118, provided below as SEQ ID NO: 6.
  • Occludin OCLN
  • NCBI accession no. AAH29886 see also UNIPROT accession no. Q16625
  • SEQ ID NO:7 An amino acid sequence for a human OCLN polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. AAH29886; see also UNIPROT accession no. Q16625) and shown below as SEQ ID NO:7.
  • the OCLN gene encodes the OCLN polypeptide with SEQ ID NO:7.
  • the OCLN gene is on chromosome 5 (location NC_000005.10 (69492547 . . . 69558104)).
  • a nucleotide sequence that encodes the OCLN polypeptide with SEQ ID NO:7 is available as NCBI accession no. NG_028291.1.
  • a cDNA sequence encoding the polypeptide having UNIPROT accession no. Q16625 is available as European Nucleotide Archive accession no. U49184, provided below as SEQ ID NO:8.
  • NCBI accession no. NP_067018 See also UNIPROT accession no. P56747.2
  • SEQ ID NO:13 An amino acid sequence for a human claudin-6 (CLDN6) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. NP_067018; see also UNIPROT accession no. P56747.2) and shown below as SEQ ID NO:13.
  • the CLDN6 gene encodes the CLDN6 polypeptide with SEQ ID NO:13.
  • the CLDN6 gene is on chromosome 16 (location NC_000016.10 (3014712 . . . 3018183, complement)).
  • a cDNA sequence that encodes the CLDN6 polypeptide with SEQ ID NO: 13 is available as NCBI accession no. NM_021195.5, shown below as SEQ ID NO: 14.
  • the CLDN7 gene encodes the CLDN7 polypeptide with SEQ ID NO:15.
  • the CLDN7 gene is on chromosome 17 (NC_000017.11 (7259903 . . . 7263213, complement).
  • a cDNA sequence that encodes the CLDN7 polypeptide with SEQ ID NO: 15 is available as NCBI accession no. NM_001307.6, shown below as SEQ ID NO:16.
  • Zonula occludens, OCLN, and claudin (CLDN) sequences can vary amongst the human population. Variants can include codon variations and/or conservative amino acid changes. Zonula occludens (TJP), OCLN, and claudin (CLDN) nucleotide and protein sequences can also include non-conservative variations.
  • the zonula occludens (TJP), OCLN, and claudin (CLDN) nucleic acids or proteins can have at least 85% sequence identity and/or complementary, or at least 90% sequence identity and/or complementary, or at least 95% sequence identity and/or complementarity, or at least 96% sequence identity and/or complementarity, or at least 97% sequence identity and/or complementarity, or at least 98% sequence identity and/or complementarity, or at least 99% sequence identity and/or complementarity to any of the Zonula occludens (TJP), OCLN, and claudin (CLDN) nucleic acid or protein sequences described herein.
  • tight junction gene products e.g., ZO1
  • ZO1 tight junction gene products
  • Loss of function modifications to tight junction genes and gene products can be introduced by any method.
  • Other possible methods of silencing/disrupting tight junction genes include using short interfering RNA (siRNA), using CRISPR to knockout or mutate a tight junction gene, or simply using chemical inhibition (EDTA or other calcium chelators, for example).
  • genetic loci encoding tight junction proteins can be modified in human iPSC lines by deletion, insertion, or substitution.
  • a variety of methods and inhibitors can be used to reduce the function of these tight junction proteins.
  • the hiPSCs or iMeLCs can be contacted with CRISPRi ribonucleoprotein (RNP) complexes, inhibitory nucleic acids, expression vectors, virus-like particles (VLP), CRISPR-related, and combinations thereof that target the tight junction genes or mRNAs.
  • RNP CRISPRi ribonucleoprotein
  • VLP virus-like particles
  • the Cas system can recognize any sequence in the genome that matches 20 bases of a gRNA.
  • each gRNA should also be adjacent to a “Protospacer Adjacent Motif” (PAM), which is invariant for each type of Cas protein, because the PAM binds directly to the Cas protein.
  • PAM Protospacer Adjacent Motif
  • the guide RNAs can have a PAM site sequence that can be bound by a Cas protein.
  • nucleases and systems that can be used for gene editing.
  • the nuclease employed can in some cases be any DNA binding protein with nuclease activity.
  • nuclease include Streptococcus pyogenes Cas (SpCas9) nucleases, Staphylococcus aureus Cas9 (SpCas9) nucleases, Francisella novicida Cas2 (FnCas2, also called dFnCpf1) nucleases, Zinc Finger Nucleases (ZFN), Meganuclease, Transcription activator-like effector nucleases (TALEN), Fok-I nucleases, any DNA binding protein with nuclease activity, any DNA binding protein bound to a nuclease, or any combinations thereof.
  • the CRISPR-Cas systems are generally the most widely used.
  • the nuclease is therefore a Cas nuclease.
  • CRISPR-Cas systems are generally divided into two classes.
  • the class 1 system contains types I, III and IV, and the class 2 system contains types II, V, and VI.
  • the class 1 CRISPR-Cas system uses a complex of several Cas proteins, whereas the class 2 system only uses a single Cas protein with multiple domains.
  • the class 2 CRISPR-Cas system is usually preferable for gene-engineering applications because of its simplicity and ease of use.
  • Cas nucleases can be employed in the methods described herein. Three species that have been best characterized are provided as examples. The most commonly used Cas nuclease is a Streptococcus pyogenes Cas9, (SpCas9). More recently described forms of Cas include Staphylococcus aureus Cas9 (SaCas9) and Francisella novicida Cas2 (FnCas2, also called FnCpf1).
  • SpCas9 Streptococcus pyogenes Cas9
  • More recently described forms of Cas include Staphylococcus aureus Cas9 (SaCas9) and Francisella novicida Cas2 (FnCas2, also called FnCpf1).
  • Inhibitory nucleic acids can be used to reduce the expression and/or translation of tight junction. Such inhibitory nucleic acids can specifically bind to tight junction nucleic acids, including nascent RNAs, that encode a tight junction protein. Anti-sense oligonucleotides have been used to silence regulatory elements as well.
  • An inhibitory nucleic acid can have at least one segment that will hybridize to tight junction nucleic acid under intracellular or stringent conditions.
  • the inhibitory nucleic acid can reduce processing, expression, and/or translation of a nucleic acid encoding tight junction.
  • An inhibitory nucleic acid may hybridize to a genomic DNA, a messenger RNA, nascent RNA, or a combination thereof.
  • An inhibitory nucleic acid may be incorporated into a plasmid vector or viral DNA. It may be single stranded or double stranded, circular, or linear.
  • An inhibitory nucleic acid can be a polymer of ribose nucleotides (RNAi) or deoxyribose nucleotides having more than 13 nucleotides in length.
  • An inhibitory nucleic acid may include naturally-occurring nucleotides; synthetic, modified, or pseudo-nucleotides such as phosphorothiolates; as well as nucleotides having a detectable label such as P 32 , biotin or digoxigenin.
  • An inhibitory nucleic acid can reduce the expression, processing, and/or translation of a tight junction nucleic acid.
  • Such an inhibitory nucleic acid may be completely complementary to a segment of tight junction nucleic acid (e.g., a tight junction mRNA or tight junction nascent transcript).
  • a segment of tight junction nucleic acid e.g., a tight junction mRNA or tight junction nascent transcript.
  • An inhibitory nucleic acid can hybridize to a tight junction nucleic acid under intracellular conditions or under stringent hybridization conditions and is sufficient to inhibit expression of a tight junction nucleic acid.
  • Intracellular conditions refer to conditions such as temperature, pH and salt concentrations typically found inside a cell, e.g. a target cell described herein.
  • Inhibitory nucleic acids of the invention include, for example, a short hairpin RNA, a small interfering RNA, a ribozyme, or an antisense nucleic acid molecule.
  • the inhibitory nucleic acid molecule may be single (e.g., an antisense oligonucleotide) or double stranded (e.g., a siRNA) and may function in an enzyme-dependent manner or by steric blocking.
  • Inhibitory nucleic acid molecules that function in an enzyme-dependent manner include forms dependent on RNase H activity to degrade target mRNA. These include single-stranded DNA, RNA, and phosphorothioate molecules, as well as the double-stranded RNAi/siRNA system that involves target mRNA recognition through sense-antisense strand pairing followed by degradation of the target mRNA by the RNA-induced silencing complex.
  • Steric blocking inhibitory nucleic acids which are RNase-H independent, interfere with gene expression or other mRNA-dependent cellular processes by binding to a target mRNA and getting in the way of other processes.
  • Steric blocking inhibitory nucleic acids include 2′-O alkyl (usually in chimeras with RNase-H dependent antisense), peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholino antisense.
  • siRNAs Small interfering RNAs
  • SiRNAs may be used to specifically reduce tight junction processing or translation such that production of the encoded polypeptide is reduced.
  • SiRNAs mediate post-transcriptional gene silencing in a sequence-specific manner. See, for example, website at invitrogen.com/site/us/en/home/Products-and-Services/Applications/mai.html.
  • siRNA can mediate cleavage of the homologous endogenous mRNA transcript by guiding the complex to the homologous mRNA transcript, which is then cleaved by the complex.
  • the siRNA may be homologous to any region of the tight junction mRNA transcript.
  • the region of homology may be 50 nucleotides or less, 30 nucleotides or less in length, such as less than 25 nucleotides, or for example about 21 to 23 nucleotides in length.
  • SiRNA is typically double stranded and may have two-nucleotide 3′ overhangs, for example, 3′ overhanging UU dinucleotides. Methods for designing siRNAs are available, see, for example, Elbashir et al. Nature 411:494-498 (2001); Harborth et al. Antisense Nucleic Acid Drug Dev. 13:83-106 (2003).
  • the pSuppressorNeo vector for expressing hairpin siRNA can be used to make siRNA or shRNA for inhibiting tight junction expression.
  • the construction of the siRNA or shRNA expression plasmid involves the selection of the target region of the mRNA, which can be a trial-and-error process.
  • Elbashir et al. have provided guidelines that appear to work ⁇ 80% of the time.
  • Elbashir, S. M., et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs . Methods, 2002. 26 (2): p. 199-213.
  • a target region may be selected preferably 50 to 100 nucleotides downstream of the start codon.
  • the 5′ and 3′ untranslated regions and regions close to the start codon should be avoided as these may be richer in regulatory protein binding sites.
  • siRNA can begin with AA, have 3′ UU overhangs for both the sense and antisense siRNA strands, and have an approximate 50% G/C content.
  • An example of a sequence for a synthetic siRNA or shRNA is 5′-AA (N19) UU, where N is any nucleotide in the mRNA sequence and should be approximately 50% G-C content.
  • the selected sequence(s) can be compared to others in the human genome database to minimize homology to other known coding sequences (e.g., by Blast search, for example, through the NCBI website).
  • Inhibitory nucleic acids may be chemically synthesized, created by in vitro transcription, or expressed from an expression vector or a PCR expression cassette. See, e.g., website at invitrogen.com/site/us/en/home/Products-and-Services/Applications/rai.html.
  • the insert encoding the siRNA may be expressed as an RNA transcript that folds into an siRNA hairpin or a shRNA.
  • the RNA transcript may include a sense siRNA sequence that is linked to its reverse complementary antisense siRNA sequence by a spacer sequence that forms the loop of the hairpin as well as a string of U's at the 3′ end.
  • the loop of the hairpin may be of any appropriate lengths, for example, 3 to 30 nucleotides in length, or about 3 to 23 nucleotides in length, and may include various nucleotide sequences including for example, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC.
  • SiRNAs also may be produced in vivo by cleavage of double-stranded RNA introduced directly or via a transgene or virus. Amplification by an RNA-dependent RNA polymerase may occur in some organisms.
  • an inhibitory nucleic acid such as a short hairpin RNA siRNA or an antisense oligonucleotide may be prepared using methods such as by expression from an expression vector or expression cassette that includes the sequence of the inhibitory nucleic acid. Alternatively, it may be prepared by chemical synthesis using naturally-occurring nucleotides, modified nucleotides, or any combinations thereof. In some embodiments, the inhibitory nucleic acids are made from modified nucleotides or non-phosphodiester bonds, for example, that are designed to increase biological stability of the inhibitory nucleic acid or to increase intracellular stability of the duplex formed between the inhibitory nucleic acid and the target tight junction nucleic acid.
  • Primordial germ cells can be differentiated into mature germ cells, including functional oocyte and sperm by in vitro culture or by implantation in a selected subject.
  • a variety of differentiation methods can be used including those described in U.S. patent application No. 20180251729.
  • Previous studies in mice illustrate methods for generating functional male and female gametes from PGCLCs in vivo, which can then be used to produce live offspring through IVF (Hayashi et al., Cell 2011) (Hayashi et al., Science 2013) (Zhou et al., Science 2013).
  • ROCK inhibitor Y-276932 (10 uM; Selleckchem) was added to the media to promote cell survival after passaging. All generated cell lines were karyotyped prior to expansion and confirmed as normal cells both by Cell Line Genetics and by using the hPSC Genetic Analysis Kit (Stem Cell Technologies Cat. #07550). The cells were also regularly tested for mycoplasma using a MycoAlert Mycoplasma Detection Kit (Lonza).
  • ZO1-WTC line To generate the ZO1-WTC line, four CRISPRi gRNAs were designed to bind within 150 bp of the transcription start site of ZO1 and cloned into the gRNA-CKB vector at the BsmB1 restriction site, following the protocol described in Mandegar et al. (2016).
  • the sequences of the ZO1 guide RNAs that were used are shown in Table 2 below.
  • Vectors containing each gRNA sequence were individually nucleofected into the WTC-LMNB1-mEGFP line (containing the CRISPRi-KRAB construct) using the Human Stem Cell Nucleofector Kit 1 solution with the Amaxa nucleofector 2b device (Lonza). Nucleofected cells were subsequently seeded at a density of 8,000 cells/cm 2 and recovered in mTESR1 media supplemented with ROCK inhibitor Y-276932 (10 uM) for two days. Guide selection was performed with blasticidin (10 ug/mL, ThermoFisher Scientific) for seven days, and clonal populations were generated through colony picking.
  • Knockdown efficiency was evaluated through exposure to doxycycline (2 uM) for five days, after which mRNA was isolated, and relative levels of ZO1 were assessed through qPCR. Levels of ZO1 were normalized to copy numbers from the same line without CRISPRi induction.
  • ZWT ZWT, ⁇ DOX
  • ZKD ZKD, +DOX
  • BMP-4 50 ng/mL
  • the ZO1 knockdown cells were then stained for appropriate germ lineage markers. Note that for these experiments involving evaluation of the ability of monolayers and cell colonies to form PGCLCs, only ZO1 knockdown cells were used (because wild type cells in monolayers and colonies do not form PGCLCs without basolateral exposure to BMP).
  • Uniform colonies ( ⁇ 100 ZO1 KD cells/colony) were achieved by seeding about 10,000 cells in mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM) from each condition into 400 by 400 mm PDMS microwell inserts (containing approximately 975 microwells) and force aggregating the cells through centrifugation at 200RCF for 3 minutes, using protocols adapted from those by Hookway et al., 2016; Ungrin et al., 2008 ( FIG. 2 B ). After 18 hours, the aggregates were transferred in mTESR1 to Matrigel-coated 96 well plates at a density of approximately 10 aggregates/well. The cells were then allowed to attach and flatten into two dimensional (2D) colonies over the course of 24 hours prior to stimulation with BMP-4.
  • ROCK inhibitor Y-27632 10 uM
  • ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells were seeded in mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM) into 96 well plates at a density between 100-350 cells/mm 2 .
  • the cells were induced with BMP-4 (50 ng/ml) in mTESR1.
  • BMP-4 50 ng/ml
  • the cells were fixed prior to staining for PGCLC and other somatic lineage markers.
  • mRNA was collected from the 48 hour timepoint for qPCR analysis, the primers used for qPCR are listed in Tables 3-4.
  • Corning Costar Transwell plates with a 6.5 mm diameter and 0.4 ⁇ m pore size were used. Transwell membranes were coated overnight with Matrigel. Prior to seeding, the Matrigel was removed and the membrane was rinsed 3 ⁇ with PBS+/+ and then put into mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM). Cells were then immediately seeded onto the transwell membranes at a density of 500-1,500 cells/mm 2 (16,600-49,800 cells/well). Twenty-four hours later, ROCK inhibitor was removed, and the cells were fed with fresh mTESR1.
  • BMP-4 was added to both the apical (top) and basolateral (bottom) compartments. Forty-eight hours after BMP-4 induction, the transwells were fixed prior to staining for PGCLC and other somatic lineage markers ( FIG. 3 ). Prior to imaging, the transwell membrane was removed and mounted onto a glass coverslip. 10 ng/mL BMP4 in transwells with a cell density of 750-1,000 cells/mm2 was optimal for PGCLC induction.
  • PSCs were first force aggregated into 400 ⁇ 400 mm PDMS microwell inserts (24-well plate sized, ⁇ 975 microwells/insert) using previously published protocols (Libby et al., bioRxiv 1-23 (2016); Hookway et al., Methods 101, 11-20 (2016); Ungrin et al., PLOS One 3, (2008)).
  • PSCs were dissociated, resuspended in mTESR1 supplemented with ROCK inhibitor (10 uM), seeded into the microwell inserts at a concentration of ⁇ 50-100 cells/well, centrifuged at 200 relative centrifugal field (rcf) for 3 minutes, and left overnight to condense into aggregates.
  • the aggregates ( ⁇ 50-100 cells in size) were resuspended in mTESR1 supplemented with ROCK inhibitor (10 uM) and transferred to Matrigel-coated 96 well plates at a concentration of approximately ⁇ 15 aggregates/well, where they were allowed to attach and form 2D colonies. After 24 hours, ROCK inhibitor was removed and the colonies were fed with mTESR1.
  • Corning Costar Transwell plates with a 6.5 mm diameter and 0.4 ⁇ m pore size were used. Transwell membranes were coated overnight with Matrigel. Prior to seeding, the Matrigel was removed and the membrane was rinsed 3 ⁇ with PBS+/+ and then put into mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM). Cells were then immediately seeded onto the transwell membranes at a density of 1,500 cells/mm 2 (49,800 cells/well). 24 hours later the ROCK inhibitor was removed, and the cells were fed with fresh mTESR1.
  • the membranes were imaged on an EVOS fluorescence microscope at 10 ⁇ to visualize whether the GFP labelled cellular nuclei reached confluence and were completely covering the membrane.
  • the inventors had previously determined that this protocol generates intact epithelia at this timepoint.
  • BMP4 50 ng/ml was added to either the apical (top) or basolateral (bottom) compartments of the transwell.
  • the transwells were fixed at the appropriate time points by transferring the insert to a new 24 well plate, rinsing with PBS, and fixing with 4% PFA.
  • FITC conjugated to 40-kDa dextran (Sigma-Aldrich) was added to the apical compartment and 10 ul of media was collected from basolateral compartment at various timepoints, which was mixed with 90 ul of PBS onto a 96-well dark-sided plate. After the time course was completed, a plate reader was used to take fluorescence measurements of our samples over time.
  • ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells were seeded at a density of 250 cells/mm 2 onto standard culture 6-well plates in mTESR1 supplemented with ROCK inhibitor (10 uM). 24 hours later, ROCK inhibitor was removed, and the cells were fed with fresh mTESR1. 24 hours after ROCK inhibitor removal, cell lysates for the pluripotent condition were prepared by putting 1.5 mL RLT (lysis) buffer/well for 3 minutes, and freezing this lysate at ⁇ 80° C. for subsequent RNA extraction. Simultaneously, BMP4 (50 ng/ml) was added to the differentiated condition.
  • RNA extraction was performed using Qiagen's RNBasy kit, and samples were subsequently shipped to Novogene for library preparation and sequencing (Illumina, PE150, 20M paired reads).
  • ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells were seeded and cultured as described in the RNA sequencing section. Only pluripotent samples were sent for sequencing. To do this, cells were dissociated using Accutase and resuspended in 200 ul PBS+proteinase K, and then frozen at ⁇ 20° C. for subsequent DNA extraction. DNA extraction was performed using Qiagen's DNA extraction kit. Samples were subsequently sent to CD Genomics for whole genome bisulfite sequencing (Illumina, PE150, 250M paired reads).
  • This Example illustrates generation of primordial germ-like cells (PGCLCs) from hiPSC cells modified to knockdown ZO1.
  • PPCLCs primordial germ-like cells
  • a doxycycline (DOX)-inducible CRISPR interference system was made for integration into the WTB (female) and WTC (male) parent hiPSC lines ( FIG. 1 A ).
  • the CRISPR interference system was comprised of two components: a dCas9KRAB repressor driven by a TetO promoter that was inserted into the AAVS1 safe harbor locus and that is expressed only under DOX treatment, and a constitutively expressed guide RNA (gRNA) that targets the transcriptional start site of the ZO1 gene.
  • the ZO1-specific gRNA (Table 2; FIG. 1 A ) was encoded in a randomly-integrating plasmid that also expressed a blasticidin selection gene.
  • DOX-inducible expression of Cas9 enabled temporal control of its gene expression.
  • These constructs were transfected into both the WTB and WTC hiPSC CRISPRi cell lines.
  • Knockdown of ZO1 was achieved after 5 days of DOX treatment in cells cultured in mTESR on Matrigel coated plates (seeding density 120 cells/mm 2 ). The cells were passaged every three days using Accutase for cell displacement.
  • hPGCLC induction was commenced by adding 50 ng/mL BMP4 directly to a monolayer of ZO1 knockdown hiPSCs at seeding densities between (100-2000 cells/mm 2 ) for at least two days.
  • FIG. 1 D show that ZO1 knockdown results in loss of tight junction barrier function as measured by FITC-Dextran diffusion. Hence, apical to basolateral diffusion is disrupted by ZO1 knockdown.
  • FIG. 2 B illustrates methods tested for generating PGCLCs from pluripotent stem cells.
  • Knock down (KD) of ZO1 expression is not necessary for generating PGCLCs when BMP4 is provided basolaterally in a culture medium such as mTESR ( FIG. 2 B top row with BMP4 on the bottom row).
  • ZO1 knockdown (KD) can be used to facilitate PGCLC generation by DOX-induced KD ( FIG. 2 B , middle row).
  • Addition of BMP4, especially basolateral addition of BMP4, to ZO1 knockdown PSCs can also generate PGCLCs.
  • Example 4 BMP Pathway Activation Correlates with Regional Loss of ZO1
  • this phenotype is also present in micropatterned colonies, regions of high density lose ZO1 and overlap with pSMAD1 activation upon BMP4 stimulation ( FIG. 5 F ). Discrepancies in previously reported pSMAD1 pre-patterns may therefore be explained in part to regional changes in density and consequent effects on epithelial structure.
  • ZO1 expression inversely correlates with pSMAD1 activation even in the context of unconfined colonies with uniform density. For example, at early timepoints upon induction with BMP4, pSMAD1 activity is largely limited to the edge of colonies. ZO1 expression does not fully extend to the edge of the colony, and tapers off a distance of approximately one cell layer before reaching the edge.
  • Example 5 ZO1 Knockdown Leads to Ubiquitous and Sustained Pathway Activation
  • CRISPRi DOX inducible CRISPR interference
  • RNA and protein expression are significantly depleted after five days of DOX treatment, as shown by qPCR, immunofluorescence (IF), and western blot ( FIGS. 1 B- 1 C, 6 B ).
  • Most of the characterization in the WTC ZO1 CRISPRi line was performed with and without DOX (referred to in the text as ZO1 wild type (ZWT) and ZO1 knockdown (ZKD), respectively), however, the results for the WTB ZO1 CRISPRi line were phenotypically similar and reproducible.
  • ZO1 knockdown cells grew in somewhat denser colonies and exhibited rounder nuclear shapes ( FIG. 6 C- 6 D ). Where ZO1 wild type nuclei are stretched and flat, ZO1 knockdown nuclei are taller and more rounded, likely as a result of severed connections between the cell-cell junctions and the actin cytoskeleton/nuclear lamina.
  • ZO1 wild type When grown as unconfined colonies and exposed to BMP4, ZO1 wild type largely limited pSMAD1 expression to the colony edge at early timepoints (15 min-1 hr) ( FIG. 5 C -SD). At later timepoints (6 hours), pSMAD1 is detectable in cells located centrally within the colony. However, pSMAD1 expression is subject to inhibitor feedback loops. Thus, this pathway activation is shut off by 48 hours in ZO1 wild type cells ( FIG. 6 E- 6 F ). Strikingly, at early timepoints, the ZO1 knockdown colonies displayed pSMAD1 throughout the colony ( FIG. 6 F ). Furthermore, ZO1 knockdown cells maintain pSMAD1 activation over time ( FIG.
  • NOGGIN is secreted apically and is trafficked transepithelially with assistance from glycoproteins on the apical surface.
  • ZO1 knockdown colonies show that ZO1 is not only important for preventing ligands such as BMP4 from accessing basolateral receptors, but may also be necessary in rendering the cells sensitive to some inhibitors, presumably by maintaining expression of the apical surface glycoproteins that enable transepithelial trafficking of apically secreted inhibitors such as NOGGIN or sequestration/concentration of other basolaterally secreted morphogen inhibitors within the colony interior.
  • This observation is reinforced by the fact that ZO1 knockdown cells also exhibit loss of apical Ezrin expression ( FIG. 1 F ), which can be important in tethering apical glycoproteins to the actin cytoskeleton.
  • ZO1 knockdown causes mixing of apical/basolateral domain elements through the plasma membrane and disrupted trafficking of receptors to their proper domains (loss of apical/basolateral polarity), or ZO1 knockdown causes increased permeability to signaling molecules (loss of barrier function).
  • ZO1 knockdown causes mixing of apical/basolateral domain elements through the plasma membrane and disrupted trafficking of receptors to their proper domains (loss of apical/basolateral polarity), or ZO1 knockdown causes increased permeability to signaling molecules (loss of barrier function).
  • the inventors first characterized apical/basolateral polarity between ZO1 wild type and ZO1 knockdown cells.
  • the Golgi apparatus faces the apical (secretory domain) direction. Therefore, the inventors evaluated positioning of the Golgi in ZO1 wild type and ZO1 knockdown cells. Z-stacks revealed that in both cell types, the Golgi sits on top of the nucleus on the apical side of the cell, suggesting that polarity of the ZO1 knockdown cells is still intact ( FIG. 7 K- 7 L ). However, staining for the apical marker Ezrin revealed significant eradication of the apical domain in ZO1 knockdown cells, characterized by punctate Ezrin localization.
  • FITC based diffusion assay was performed to look for differences in permeability in ZO1 wild type and ZO1 knockdown. Each cell type was grown on a transwell membrane and a 40 kDa dextran conjugated with FITC was added to the apical compartment ( FIG. 6 G ). The 40 kDa-FITC was selected due to its similarity in hydraulic radius to many common gastrulation-associated signaling proteins. Specifically, 40 kDa-FITC is slightly smaller than BMP4. Hence, an epithelial barrier that could exclude the 40 kDa-FITC is evidence that the epithelial barrier could also exclude BMP4.
  • the RD model proposes that in response to signal pathway activation (phosphorylation of SMAD1) by an activating species (BMP4), cells secrete more of this activator (BMP4) and its inhibitor (NOGGIN) in a feedback loop (Tewary et al., Development dev. 149658 (2017)). Differences in the diffusivities between NOGGIN and BMP4 can create a steady-state gradient of effective BMP4 concentrations across the colony, and cells sense positional information and differentiate based on both on this concentration gradient and its overlap with other members of a BMP4-induced feedback loop, including WNT and NODAL.
  • the initial pSMAD1 pre-pattern is therefore assumed to be an important indication of the shape of an RD gradient which determines the shape of subsequent gastrulation-associated patterning.
  • TBXT expression is substantially increased throughout the center of the colony ( FIG. 7 B ).
  • Many progenitor cell types express TBXT.
  • RNA sequencing was performed on pluripotent and 48-hour BMP4 treated ZO1 wild type and ZO1 knockdown cells.
  • hPSCs are first reset from a primed to a na ⁇ ve pluripotency state, as primed hPSCs are thought to have lost the developmental potential to generate PGCLCs.
  • iMeLC or na ⁇ ve pluripotency pre-induction both protocols failed to efficiently generate PGCLCs, providing only about 1-2% efficiency of generating PGCLCs.
  • ZO1 knockdown cells do not undergo any form of pre-induction yet are able to produce a robust PGCLC population.
  • RNA sequencing showed that aside from ZO1 and ZNF10 (which is part of the CRISPRi machinery), few genes are both significantly and substantially differentially expressed between ZO1 wild type and ZO1 knockdown cells ( FIG. 8 G ), and no significant changes are shown in major canonical pluripotency markers ( FIG. 8 C ).
  • Whole genome bisulfite sequencing shows that while several probes are differentially methylated ( FIG. 8 D, 8 H ), there are no global changes in methylation of probes between ZO1 wild type and ZO1 knockdown cells, which would be expected if a resetting process occurred.
  • ZO1 knockdown cells are predisposed to PGCLC fates because, unlike ZO1 wild type cells which undergo NOGGIN-related BMP4-pathway inhibition at later timepoints, ZO1 knockdown cells are able to maintain BMP4-pathway activation.
  • RNA sequencing of stimulated ZO1 wild type and ZO1 knockdown cells grown on transwells showed remarkable similarities in marker expression between the two samples, demonstrating that most of the observed changes in cell fate are a direct result of increased signal pathway activation.
  • the total number of differentially expressed genes between ZO1 wild type and ZO1 knockdown samples was significantly higher in standard culture (3150) versus in transwell (35) culture, highlighting the magnitude of the expression changes dependent solely on changes in pSMAD1 signaling. Of these 35 genes, unbiased clustering and GO analysis demonstrated that ZO1 knockdown cells still have a slight bias towards mesendodermal lineages, as illustrated in Table 7 below.
  • a system comprising pluripotent stem cells supported on a porous surface in a culture medium that contains BMP.
  • pluripotent stem cells are human pluripotent stem cells.
  • the tight junction gene is at least one endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • the chelator or chemical inhibitor is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis ( ⁇ -aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), dimercaptosuccinic acid, dimercaprol, genistein, 1-tert-Butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2), glycyrrhizin, or a combination thereof.
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethylene glycol-bis ( ⁇ -aminoethyl ether)-N,N,N′,N′-tetraacetic acid
  • dimercaptosuccinic acid dimercaprol
  • genistein 1-tert-Butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimi
  • any one of statements 23-36, wherein inhibiting the tight junction formation comprises incubating the population of pluripotent stem cells with PTPN1, acetylaldehyde, genistein, protein phosphatase 2 (PP2), Clostridium perfringens enterotoxins (and their derived mutants), monoclonal antibodies against Claudin-1 (75A, OM-7D3-B3, 3A2, 6F6), monoclonal antibodies against Claudin-6 (IMAB027), Claudin-2 (1A2), monoclonal antibodies against Claudin-5 (R9, R2, 2B12), monoclonal antibodies against Occludin (1-3, 67-2), and combinations thereof.
  • PTPN1 acetylaldehyde
  • genistein protein phosphatase 2
  • PP2 protein phosphatase 2
  • Clostridium perfringens enterotoxins and their derived mutants
  • monoclonal antibodies against Claudin-1 75A, OM-7
  • inhibiting the tight junction formation comprises inhibiting expression or function of at least one endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • ZO1 endogenous zonula occludens-1
  • ZO2 zonula occludens-2
  • ZO3 zonula occludens-3
  • inhibiting the tight junction formation comprises inhibiting expression or function of at least one endogenous zonula occludens-1 (ZO1) allele.
  • pluripotent stem cells are human pluripotent stem cells.
  • the BMP is at a concentration of at least 0.1 ng/ml, or at least 1 ng/ml, or at about 2 ng/ml or at least 5 ng/ml, or at least 10 ng/ml, or at least 20 ng/ml, or at least 25 ng/ml, or at least 30 ng/ml, or at least 35 ng/ml, or at least 40 ng/ml, or at least 50 ng/ml.
  • a modified pluripotent stem cell comprising a knockdown or knockout of an endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • a population of modified pluripotent stem cells each primordial germ cell comprising a knockdown or knockout of an endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • ZO1 endogenous zonula occludens-1
  • ZO2 zonula occludens-2
  • ZO3 zonula occludens-3
  • nucleic acid or “a protein” or “a cell” includes a plurality of such nucleic acids, proteins, or cells (for example, a solution or dried preparation of nucleic acids or expression cassettes, a solution of proteins, or a population of cells), and so forth.
  • the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.

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Abstract

Described herein are compositions, systems, and methods for obtaining primordial germ cells (PGCs) from pluripotent stem cells (PSCs). Inhibiting or bypassing tight junction formation in a population of pluripotent stem cells to generate a modified cell population, and contacting the modified cell population with BMP. Where inhibiting or bypassing tight junction formation includes incubating the population of pluripotent stem cells.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a U.S. National Stage Filing under 35 U.S.C. 371 from International Patent Application Serial No. PCT/US2022/034869, filed Jun. 24, 2022, Published as WO2022/272042 on Dec. 29, 2022, which application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 63/214,901 entitled “Human Primordial Germ Cells from Human Induced Pluripotent Stem Cells,” filed Jun. 25, 2021, the complete disclosures of which are incorporated herein by reference in their entireties.
    • GOVERNMENT SUPPORT
  • This invention was made with government support under CBET 0939511 awarded by the National Science Foundation. The government has certain rights in the invention.
    • INCORPORATION BY REFERENCE OF SEQUENCE LISTING
  • This application contains a Sequence Listing which has been submitted electronically in ST25 format and hereby incorporated by reference in its entirety. Said ST256 file, created on Jul. 3, 2024, is name 3730194US1.txt and is 80,536 bytes in size.
    • BACKGROUND
  • Human primordial germ cells (PGCs) are the precursors to human male and female sex cells (spermatozoa and oocytes). Ethical considerations largely prevent close interrogation of the development and specification of primordial germ cells in a human embryo. If primordial germ cells could be generated in vitro they could be used to differentiate functional oocytes and spermatozoa that could be used for In Vitro Fertilization (IVF), which would address a range of problems that currently plague IVF treatments such as: low retrieval of oocytes, ovarian hyperstimulation syndrome (which occurs during the hormone treatments to retrieve the oocytes), and senescence of sex cell production for older couples.
  • Embryonic pluripotent stem cells (PSCs) are taken directly from the inner cell mass/epiblast of a human embryo. Induced PSCs are reprogrammed from somatic cells taken from a patient (through methods such as a skin biopsy, blood draw, cheek swab, etc.). Typically, when these embryonic or induced PSCs are cultured in vitro, they form a polarized epithelial “barrier” structure and are considered “primed.” Primed PSCs structurally, transcriptionally, and epigenetically resemble post-implantation/pre-gastrulation (E9-E12) pluripotent stem cells in the epiblast and have the potential to form any somatic cell type (lungs, heart, kidney, skin, etc.) found in the body, if they are exposed to the correct differentiation cues.
  • However, researchers generally believe that cultured primed PSCs do not have the ability to form primordial germ cells (PGCs), which are the precursors to sperm and ova, because primed PSCs are thought to be too committed at this stage to a somatic developmental trajectory. Hence, currently available methods for generating primordial germ cells (PGCs) typically involve chemical treatments and/or genetic modifications to revert the primed PSCs to a more naïve state, followed by use of a several factors to induce differentiation into primordial germ cells (PGCs).
    • SUMMARY
  • Described herein are systems, compositions, and methods for obtaining primordial germ cells (PGCs) from pluripotent stem cells (PSCs). For example, the pluripotent stem cells employed can be human induced pluripotent stem cells (hiPSCs)). The PSCs can be genetically modified (e.g., to repair genetic mutations or to facilitate PGC differentiation). In some cases, the PSCs can be genetically modified to express genes involved in PGC specification or genetically modified to make the PSCs more susceptible to PGC differentiation.
  • However, as described herein, such genetic modification is not needed to produce primordial germ cells from PSCs. Instead, an effective method is described herein that involves basolateral stimulation of human induced pluripotent stem cells with BMP. For example, the methods can involve seeding PSCs into vessels that provide BMP with basolateral access to the PSCs.
  • PGCs are the first step to differentiating functional oocytes and spermatozoa that can be used for In Vitro Fertilization (IVF). The methods described herein allow men and women who are experiencing fertility problems to undergo a simple cell retrieval (e.g., a simple skin biopsy), followed by reprogramming of their cells into hiPSCs and differentiation of the hiPSCs into PGCs. The PGCs can then be differentiated into functional sex cells. Use of such iPSC-derived PGCs addresses a range of problems that currently plague IVF treatments, such as: low retrieval of oocytes, ovarian hyperstimulation syndrome (which occurs during the hormone treatments to retrieve the oocytes), and senescence of sex cell production for older couples. Additionally, simple and non-invasive PGC derivation facilitates screening of genetic disease for at-risk couples, enabling trans-differentiation and IVF of sex cells for same sex couples. Some beneficial products and methods provided are:
      • 1. Minimally invasive and hormone-free oocyte retrieval:
        • a. No physician monitoring is needed,
        • b. No expensive hospital visits/hormone treatments are needed,
        • c. Cheaper and more efficient derivation of PGCs and oocytes.
      • 2. Derivation of oocytes and spermatozoa from older patients with traditionally less sex cell production viability.
      • 3. Expanded and biopsy-free screening for genetic disease.
      • 4. Trans-differentiation of PGCs to oocytes/spermatozoa of opposite sex.
  • Methods and systems are described herein that are useful for generating primordial germ cells. Such methods can involve reducing or bypassing barrier function in a population of pluripotent stem cells to generate modified cell population and contacting the modified cell population with BMP. For example, the methods can involve inhibiting or bypassing tight junction formation in a population of pluripotent stem cells to generate a modified cell population, and contacting the modified cell population with BMP. As used herein, “inhibiting tight junction(s)” means reducing the incidence of tight junction formation, maintaining pluripotent stem cells in a naïve state, and/or bypassing tight junction formation. Inhibiting or bypassing tight junction formation can include:
      • a. incubating the population of pluripotent stem cells on a porous surface to bypass apical tight junctions;
      • b. contacting the population of pluripotent stem cells with one or more inhibitory nucleic acids that bind one or more tight junction nucleic acids (one or more tight junction mRNA or DNA);
      • c. contacting the population of pluripotent stem cells with one or more CRISPRi ribonucleoprotein (RNP) complexes targeted to one or more tight junction gene;
      • d. contacting the population of pluripotent stem cells with one or more expression vectors or virus-like particles (VLP) encoding one or more guide RNAs that can bind one or more tight junction gene;
      • e. contacting the population of pluripotent stem cells with one or more chelators (e.g., calcium chelators) or chemical inhibitors; or
      • f. combinations thereof.
        The modified cell population is modified relative to a control cell population that has not be treated or manipulated to inhibit or bypass tight junction formation.
  • In some cases pluripotent stem cells can be supported on a porous surface in a culture medium that contains BMP. This method does not require genetic modification of the pluripotent stem cells to provide primordial germ cells. The porous surface can be a membrane that freely allows nutrients and morphogens (e.g., proteins such as BMP) to circulate through the membrane. One type of culture apparatus that includes a porous surface for culture of the cells is a transwell culture system. Examples of materials that can be used for the porous surface include porous polycarbonate, polyester (PET), and/or collagen-coated polytetrafluoroethylene (PTFE) materials.
  • The pluripotent stem cells can be induced pluripotent stem cells (iPSCs), such as human induced pluripotent stem cells (hiPSCs). Cells can be obtained from a selected subject, iPSCs can be generated from the subject's cells, and those iPSCs can then be converted into primordial germ cells. Mature germ cells can be generated from the primordial germ cells and used for in vitro fertilization to provide an embryo that can be implanted for gestation in a female. Hence, the pluripotent stem cells or the induced pluripotent stem cells can be autologous or allogenic to a subject who desires in vitro fertilization. The subject can be any mammalian or avian subject. In addition to human subjects, the methods and systems can be used to provide primordial germ cells for domesticated animals, wild animal species, endangered animal species (e.g., an animal on an endangered species list), as well as animal species that are extinct or are in danger of becoming extinct.
  • The pluripotent stem cells can be genetically modified. For example, the pluripotent stem cells can be genetically modified to correct a genetic defect.
  • In some cases the pluripotent stem cells can be genetically modified to reduce the expression or function of an endogenous tight junction gene. For example, such a tight junction gene can be at least one endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene. At least one tight junction allele of any of these genes can be genetically modified. In some cases, two tight junction alleles of any of these genes can be genetically modified.
  • The BMP used in the system can be BMP2, BMP4, or a combination thereof.
  • Also described herein are methods that involve incubating one or more pluripotent stem cells on a porous surface within a system comprising in a culture medium that contains BMP. The pluripotent stem cells can be induced pluripotent stem cells (iPSCs), such as human induced pluripotent stem cells (hiPSCs). The pluripotent stem cells can be genetically modified. For example, the pluripotent stem cells can be genetically modified to correct a genetic defect.
  • The methods and systems described herein can involve culturing cells on porous surfaces (e.g., a transwell) under conditions that provide growth of the cells. Such a porous surface (e.g., transwell) can have an apical compartment as well as a basolateral compartment. The pluripotent stem cells can be one the porous surface in the apical compartment and receive BMP from at least a basolateral compartment.
  • The conditions used for generating PGCs can include culturing the cells at temperatures above 30° C., or above 33° C., or above 35° C., or above 36° C. The temperature should be below 42° C., or below 40° C., or below 39° C., or below 38° C. For example, the temperature can be about 37° C. The culture medium can include a ROCK inhibitor.
    • DESCRIPTION OF THE FIGURES
  • FIG. 1A-1H illustrate knockdown of zonula occludens-1 (ZO1) in human induced pluripotent stem cells (hiPSCs) and the functional consequences of such knockdown. FIG. 1A is a schematic illustrating the CRISPR-interference platform used to knockdown zonula occludens-1 (ZO1) in hiPSCs. Briefly, a TET-responsive dead Cas9-KRAB construct was knocked into the AAVS1 locus of the hiPSCs. dCas9-KRAB was expressed upon addition of Doxycycline (DOX). Upon constitutive expression of a ZO1 guide RNA (designed by the inventors), transcription of ZO1 was blocked. FIG. 1B shows expression of ZO1 and the nuclear marker Lamin-B1 (LMNB1) in the hiPSCs after exposure of the cells to Doxycycline (2 uM) for several days to induce knockdown of ZO1. As illustrated, by day 5, ZO1 expression was not visibly detectable in these ZO1 knockdown cells. FIG. 1C graphically illustrates the fold change of ZO1 expression after exposure of the hiPSCs to Doxycycline (2 uM) for five days to induce knockdown of ZO1. As illustrated, by day 5, ZO1 expression was substantially undetectable. FIG. 1D illustrates fluorescent measurements of media aliquots taken over time from the basolateral side of a transwell in which a wild type cell layer or a ZO1 knockdown cell layer was maintained after addition of FITC-dextran to the apical side of the transwell. As illustrated, the wild type cell layer forms a membrane that is less permeable to the FITC-dextran than is the ZO1 knockdown cell layer. This graph illustrates how barrier function and ability to preclude diffusion of molecules from one side of a cellular monolayer to the other (apical to basolateral diffusion) is disrupted by ZO1 knockdown. FIG. 1E graphically illustrates transepithelial resistance in wild type and ZO1-knockdown cells treated for 5 days with Doxycycline (2 uM), indicating loss of barrier function with ZO1 knockdown. FIG. 1F shows images of wild type and ZO1 knockdown cells immunostained for the nuclear marker Lamin-B1 (LMNB1) or for cytovillin (EZRIN), an apical polarity protein. As shown, expression of EZRIN is attenuated with ZO1 knockdown cells, indicating loss of apical/basolateral polarity. FIG. 1G shows chromosomal images illustrating the karyotype of a ZO1 WTC-LMNB1-GFP-CRISPRi (male ZO1 knockdown line). FIG. 1H illustrates karyotyping analysis of expression from chromosomal loci demonstrating that all genetically modified lines used to validate results in this study are karyotypically normal, including the ZO1 WTC-LMNB1-GFP-CRISPRi (male ZO1 knockdown line), ZO1 WTB-CRISPRi-Gen1B (female ZO1 knockdown line) and ZO1 WTC-NANOS3-mCHERRY (male ZO1 knockdown line, with PGC reporter).
  • FIG. 2A-2E illustrate the method by which PGCLCs (primordial germ like cells, designated “like” because they are generated in vitro) are generated from ZO1 wild type and ZO1 knockdown hiPSCs. FIG. 2A illustrates that as a result of impaired barrier function, ZO1 knockdown hiPSCs lose polarized response to BMP4, enabling activation of pSMAD1 when BMP4 is presented apically (apical presentation is typical in standard/non-transwell culture). FIG. 2B is a schematic illustrating methods for determining specification bias, which was used to assay the ZO1 knockdown cells in comparison to ZO1 wild type cells. FIG. 2C shows the results of the specification bias assay delineated in FIG. 2B, demonstrating that ZO1 knockdown cells have marked bias for expressing PGC markers (BLIMP1), but also expressed SOX17, CDX2, T-box transcription factor T (TBXT or T), and SOX2. Wild type cells exhibited more SOX2 expression while ZO1 knockdown cells exhibited more BLIMP1 and TBXT expression. FIG. 2D graphically illustrates qPCR data from monolayers of control cells (−DOX) and ZO1 knockdown cells (+5 days of DOX or +14 days of DOX), treated with BMP4 for 48 hours. These results demonstrate that BMP4-treated ZO1 knockdown cells exhibit significant increases in PGC transcription factors (T, SOX17, NANOS3, and BLIMP1), validating immunofluorescent staining data from FIG. 2C. FIG. 2E shows replicate immunofluorescent staining of control (−DOX) and ZO1 knockdown (+DOX) cells after treatment with BMP4 for 48 hours to detect a panel of PGC markers (BLIMP1, SOX17, and TFAP2C). Double positive staining was used to identify primordial germ cell like cells (PGCLCs; which are primordial germ cells generated in vitro). SOX2 is not a PGC marker and was shown as a negative control. In the original SOX2 was stained blue, TFAP2C was stained blue, BLIMP1 was stained red, and SOX17 was stained green.
  • FIG. 3 schematically illustrates that PGC (also called PGCLC) differentiation can be achieved via ZO1 silencing, pharmacological inhibition of ZO1, or by growth of cells on transwell membranes in the presence of BMP4. Such growth of cells on transwell membranes requires no chemical and no structural perturbation cells, and instead is mediated by basolateral stimulation by BMP. These varied methods illustrate that loss of barrier function or heightened accessibility of BMP4 to its basolateral receptors leads to high activation of the canonical BMP-SMAD1 pathway (illustrated in FIG. 2A). For comparison, a typical epithelial cell layer in culture is schematically illustrated on the left, which forms tight junctions maintained by ZO1 and which does not produce PGCs (PGCLCs) upon stimulation with BMP4.
  • FIG. 4 schematically illustrates the role of Zonula occludens-1 (ZO1, also called TJP1) within cells and how ZO1 maintains epithelial structure. ZO1 is a tight junction protein expressed in primed pluripotent stem cells in standard in vitro culture. ZO1 forms dual-purpose adhesion plaques that endow an epithelium with both barrier and partitioning functions (polarity/directionality), thereby attenuating responses to morphogen signals (such as BMP4).
  • FIG. 5A-5H illustrate that unconfined human iPSC colonies undergo radial gastrulation-like patterning with loss of ZO1 on the colony edge. FIG. 5A illustrates a method where hiPSCs were aggregated into pyramidal wells, subsequently plated, and induced with BMP4 for 48 hours. FIG. 5B illustrates that unconfined colonies of wild type hiPSCs undergo radial patterning of gastrulation-associated markers after 48 hrs of BMP4 stimulation. FIG. 5C shows immunofluorescence images of a wild type colony edge, showing loss of ZO1 and gain of pSMAD1 at the colony edge. FIG. 5D graphically illustrates quantification of ZO1 loss and pSMAD1 gain on wild type colony edges (n=3). FIG. 5E shows images of unconfined and low/high density micropatterned colonies, with a comparison of ZO1 and pSMAD1 expression in these wild type colonies. FIG. 5F shows images of unconfined wild type colonies illustrating that they maintain honeycomb ZO1 expression over time. FIG. 5G graphically illustrates cell density measurements in unconfined wild type colonies, with a projected density curve for micropatterned colonies (assuming density of 5,000 cells/mm2 upon induction with BMP4). Epithelial range, based on structure of cell-cell junction pattern, was estimated to be in the range of 3,000-10,000 cells/mm2. FIG. 5H shows images of wild type cellular monolayers illustrating ZO1 and pSMAD1 expression as a function of cell density in monolayer culture. The epithelial structure (honeycomb cell-cell junction pattern) is lost and pSMAD1 activation is increased as cell density increases.
  • FIG. 6A-6I illustrate that ZO1 knockdown (ZKD) causes ubiquitous and sustained phosphorylation of SMAD1 throughout cellular colonies over time. FIG. 6A is a schematic illustrating that CRISPRi knockdown of ZO1 increases signaling protein accessibility. FIG. 6B shows a Western blot illustrating ZO1 protein loss in the ZO1 knockdown cell lines. The WTB (female) and WTC (male) cells are parental hiPSC lines. FIG. 6C shows immunofluorescence images and brightfield images illustrating morphological differences between ZO1 wild type and ZO1 knockdown cells. FIG. 6D graphically illustrates changes in nuclear height, area, cell density, and growth rate of ZO1 wild type and ZO1 knockdown cells. FIG. 6E graphically illustrates the fraction of pSMAD1+ cells over time, normalized to expression of LMNB1 (n≥3), in populations of ZO1 wild type and ZO1 knockdown cells. FIG. 6F shows immunofluorescence images illustrating maintained and ubiquitous phosphorylation of SMAD1 in ZO1 knockdown (ZKD) cells compared to ZO1 wild type cells over the course of 48 hours. FIG. 6G is a schematic illustrating a FITC-dextran diffusion assay. ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells were cultured on a transwell plate, 40 kDa FITC was applied to the apical side, and fluorescence measurements were taken from the basolateral compartment over time. FIG. 6H graphically illustrates the fluorescence observed from the basolateral compartment over time using the method illustrated in FIG. 6G. FIG. 6I graphically illustrates transepithelial electrical resistance (TEER) measurements in ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) monolayers.
  • FIG. 7A-7N illustrate that ZO1 knockdown (ZKD) cells are biased toward differentiation into PGCs. FIG. 7A is a schematic showing the inventors' predictions regarding spatial emergence of distinct lineages arising in ZO1 wild type (ZWT; top) and ZO1 knockdown (ZKD; bottom) colonies exposed to BMP4 under a reaction diffusion (RD)/positional information (PI) patterning model. FIG. 7B shows immunofluorescence images of canonical germ lineage markers LMNB1, CDX2, SOX2, TBXT in ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells after 48 hours of stimulation with BMP4. FIG. 7C graphically illustrates the fraction of cells positive for expression of the markers shown in FIG. 7B in wild type (ZWT) and ZO1 knockdown (ZKD) cells. FIG. 7D shows a volcano plot of RNA sequencing data illustrating log fold changes of SOX2, TBXT, and CDX2. FIG. 7E graphically illustrates RNA sequencing data illustrating expression levels of canonical germ layer markers in ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells after 48 hours of stimulation with BMP4. FIG. 7F illustrates unbiased clustering of the top 16 differentially expressed genes between ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells, highlighting increases in PGC-related genes. FIG. 7G shows immunofluorescent images of LMNB1, and PGC markers BLIMP1, SOX17, TFAP2C in ZO1 wild type (ZWT) and ZO1 knockdown cells (ZKD) after 48 hours of stimulation with BMP4. FIG. 7H illustrates that pSMAD1 expression is only activated upon basolateral (top row) BMP4 stimulation in wild type ZO1 cells, but not by apical BMP4 stimulation. However, both apical and basolateral stimulation by BMP activates pSMAD1 in ZO1 knockdown (ZKD) cells. FIG. 7I graphically illustrates levels of BMP receptor expression in ZO1 wild type and ZO1 knockdown cells as observed from RNA sequencing data. The types of BMP receptors are recited along the x-axis. FIG. 7J graphically illustrates the fold change in secreted morphogens at 12 hours of BMP4 stimulation, showing significant increases in Noggin (NOG) in the ZO1 knockdown (ZKD) cells that are not seen in ZO1 wild type cells, as detected by qPCR. FIG. 7K shows images of cells illustrating the positioning of the Golgi in ZO1 wild type (left) and ZO1 knockdown (right) cells. Z-stacks revealed that in both cell types, the Golgi sits on top of the nucleus on the apical side of the cell, indicating that polarity of the ZO1 knockdown cells is still intact. FIG. 7L graphically illustrates the fluorescence intensity of immunostained Golgi as a function of the distance from the nuclear center of ZO1 wild type and ZO1 knockdown cells, indicating that the Golgi sits on top of the nucleus on the apical side of both cell types. FIG. 7M shows images of immunofluorescent-stained ZO1 wild type cells (left) and ZO1 knockdown cells (right), illustrating that ZO1 knockdown cells lost apical Ezrin expression (dark area delineated by a white dashed line). Even in regions where Ezrin is present, the Ezrin overlaps significantly with BMPR1A (a basolateral BMP receptor). FIG. 7N graphically illustrates the ratio of EZRIN: BMPR1A in ZO1 wild type and ZO1 knockdown cells. Hence, changes occur in the amounts and localization of some apical/basolateral elements in ZO1 knockdown cells compared to wild type cells.
  • FIG. 8A-8H illustrate ZO1 knockdown cells have a bias for PGC differentiation. FIG. 8A shows images of immunofluorescent-stained ZO1 wild type and ZO1 knockdown cells illustrating expression of LMNB1, BLIMP1, SOX17, TFAP2C after 48 hours and 72 hours of stimulation with BMP4. FIG. 8B graphically illustrates the percent of ZO1 wild type and ZO1 knockdown cellular nuclei that exhibit expression of the indicated PGC markers (n≥3). FIG. 8C illustrates expression of canonical pluripotency markers in ZO1 wild type and ZO1 knockdown cells prior to BMP4 stimulation. FIG. 8D illustrates methylation levels of ZO1 wild type versus ZO1 knockdown cells; the data were from whole genome bisulfite sequencing data. FIG. 8E shows images of immunofluorescent-stained cells illustrating expression of LMNB1, BLIMP1, SOX17, TFAP2C in ZO1 wild type and ZO1 knockdown cells after 48 hours and 72 hours of stimulation with BMP4 in a female hiPSC line. FIG. 8F graphically illustrates the percent of ZO1 wild type and ZO1 knockdown cellular nuclei that exhibit expression of PGC markers (n≥3) in a female hiPSC line. FIG. 8G illustrates unbiased clustering of top 16 differentially expressed genes between ZO1 wild type and ZO1 knockdown cells in the pluripotent condition. FIG. 8H illustrates probe methylation levels between ZO1 wild type and ZO1 knockdown cells gathered from whole genome bisulfite sequencing data, probes with significant differences in methylation are darkly shaded.
  • FIG. 9A-9B illustrate that ZO1 knockdown-related PGCLC bias is a product of signaling, not changes in pluripotency. FIG. 9A shows images of immunofluorescent-stained ZO1 wild type (top) and ZO1 knockdown (bottom) cells illustrating pSMAD1 expression after basolateral BMP4 stimulation for timepoints between 0-48 hrs when the cells were grown on the transwell membranes. FIG. 9B shows images of immunofluorescent-stained ZO1 wild type and ZO1 knockdown cells illustrating expression of LMNB1, BLIMP1, SOX17, TFAP2C when the cells were grown on transwell membranes with 48 hrs of bi-directional (apical and basolateral) stimulation with BMP4 at concentrations between 5-50 ng/ml.
    •  DETAILED DESCRIPTION
  • Described herein are compositions and method for obtaining primordial germ cells (PGCs) from pluripotent stem cells (PSCs), including human induced pluripotent stem cells (hiPSCs). The compositions and methods provide useful numbers of primordial germ cells (PGCs) with an efficiency of about 50-60% and without the need for three-dimensional (3D) suspension or bioreactor culturing procedures. The epithelial barrier structure of the induced pluripotent stem cells is modified by the methods described herein either during differentiation by basolateral exposure to BMP, by exposure to tight junction inhibitors, or by using CRISPR interference (CRIPSRi) to inhibit, knock down, or knockout one or more tight junction genes or tight junction proteins.
  • As mentioned above, researchers generally believe that cultured primed PSCs do not have the ability to form primordial germ cells (PGCs), which are the precursors to sperm and ova, because the primed PSCs are thought to be too committed at this stage in their developmental trajectory. Hence, currently available in vitro differentiation protocols for generating PGC-like cells (PGCLCs) involve a step that causes primed PSCs to be reverted to a more naïve state first. This step is followed by a priming step, and differentiation with the morphogens BMP4 or BMP2. For example, currently available reprogramming methods involve manipulating primed PSCs to a more naïve PSC state that structurally/transcriptionally/epigenetically resembles the apolar inner cell mass/pre-implantation epiblast (E5-E9). This has been done through transient delivery of transgenes via expression vectors or by introducing RNA, or through exposure of the primed PSCs to various cytokines/histone deacetylases, and other chemicals and/or biological molecules (e.g., LIF, SCF, EGF, Activin A, CHIR99021).
  • However, the methods described herein do not require such genetic modification or extensive exposure to multiple chemicals and biological molecules. Instead, the methods can simply involve culturing pluripotent stem cells (e.g., human induced pluripotent stem cells (hiPSCs)) in vessels that allow BMP to basolaterally contact the pluripotent stem cells for a time sufficient for the pluripotent stem cells to differentiate into primordial germ cells. Alternatively, pluripotent stem cells (e.g., human induced pluripotent stem cells (hiPSCs)) can be cultured under conditions that transiently inhibit relevant tight junction proteins, for example, by knockdown of tight junction protein expression or through pharmacological inhibition of tight junction protein functions.
  • As demonstrated herein, tight junctions are assembled via the protein ZO1. Such tight junctions are used by cells to split the cell into two “sides”: the apical side and the basolateral side. Apical refers to the outward-facing side(s) of a cell, which have more tight junctions than the basolateral side of cell. Basolateral refers to the inward-facing side(s) of a cell. When cells are cultured on a plate or surface, the apical side is the side exposed to culture media, while the basolateral side is the side facing/attached to the plate or surface of the culture vessel.
  • Tight junctions can prevent diffusion of proteins and other small molecules between these two domains, thereby acting as a barrier. Most morphogen receptors are basolateral (facing away from the media). Hence, when cells are cultured so that at least one side rests or attaches to a surface, those cells are rendered partially or completely inaccessible to signals present in the media. Although individual free floating cells may survive briefly in suspension, they do not survive for long. Cells can be cultured for a while as aggregates in suspension but the same problems exist for aggregated cells as for cells maintained on solid surfaces-tight junctions are present on the apical sides of aggregated cells. Even when aggregated cells are disassociated, the tight junctions will quickly reassemble upon reaggregation of the cells. Aggregated cells therefore have the same barrier/receptor access problems as cells cultured on solid surfaces-morphogens in the media are not taken up, or only occasionally take up, because the tight junctions on the apical surfaces block such uptake. Under standard culture conditions using culture plates, or using flasks with cells maintained in suspension, cellular differentiation is heterogeneous because stochastic signal pathway activation occurs.
  • Reducing the inhibiting tight junction formation or bypassing tight junctions or as described herein, for example by ZO1 knockdown or by basolateral stimulation (e.g., by growing cells on a transwell), provides homogeneous and sustained signal pathway activation. Such reduction/removal of tight junctions is useful because signal pathway activation in the cells can specifically be controlled. The culture methods described herein therefore optimize the PGC differentiation, providing the least expensive and fastest differentiation protocol to generate PGCs.
    • Basolateral BMP for Generating Primordial Germ Cells
  • In their developmental trajectory from naïve to primed, pluripotent stem cells within the epiblast undergo epithelialization. Epithelialization is a dramatic structural change resulting in transformation of the apolar and largely disorganized mass of naïve PSCs in the inner cell mass (ICM) or early epiblast into a flat sheet-like structure (an epithelium). However, cultured cells that are in such a sheet-like structure, or in a monolayer, are less accessible to components in the culture medium (e.g., as shown in FIG. 3-4 ). Currently available methods typically involve contacting the apical surface of cellular monolayers. However, such methods are not effective for generating primordial germ cells, due to low activation of the canonical BMP-SMAD1 pathway (FIG. 2A).
  • As described herein, primordial germ cells can be generated from human induced pluripotent stem cells (e.g., hiPSCs) by incubating the PSCs in vessels that allow BMP to basolaterally contact the PSCs. A variety of pluripotent stem cells can be used, including induced pluripotent stem cells (iPSCs), embryonic stem cells, embryonic stem cells made by somatic cell nuclear transfer (ntES cells), or embryonic stem cells from unfertilized eggs (parthenogenesis embryonic stem cells, pES cells).
  • As used herein, the apical cell surface refers to the surface of a monolayer of cells that faces the culture medium. The apical surface does not include the cell surface that contacts the culture plate or the culture vessel or that contacts an aggregated cell mass.
  • As used herein, the basolateral cell surface refers to everything below the apical surface that can freely contact cell media. Hence the basolateral cell surface does not include the sides or the surfaces upon which the cells rest or that contact a solid surface or an aggregated cell mass. When cells are grown/maintained in a monolayer, the basolateral surface does not include the base of the cells that rest on a solid surface, or where the cells are laterally in contact with each other. The cell base and the cell apical surfaces are generally on opposite sides of the cells.
  • When generating primordial germ cells using the methods described herein, the base of the PSCs can rest upon a porous surface. The porous surface supports the cells. The porous surface can have pores of any pore size so long as the cells cannot pass through the pores. An example of a pore size range that can be used is about 0.4 μm to about 8.0 μm. Such a porous surface can be a membrane.
  • For example, culture medium containing BMP can be placed in a vessel or in wells of a culture plate. A membrane (e.g., transwell insert) can then be added and the PSCs can be seeded onto the membrane (e.g., of a transwell plate compartment). The cell medium below the cells (the basolateral compartment) therefore contains BMP.
  • In some cases the membrane can be conditioned prior to use. For example, the membrane can be incubated with extracellular matrix protein (e.g., Matrigel), and the extracellular matrix protein can be removed (e.g., by aspiration) from the membrane prior to seeding the PSCs onto the membrane.
  • The PSCs can be seeded at various densities. For example, the PSCs can be seeded at cell densities of about 10 cells/mm2 to 10,000 cells/mm2, or about 100 cells/mm2 to 9,000 cells/mm2, or about 200 cells/mm2 to 8,000 cells/mm2, or about 400 cells/mm2 to 6,000 cells/mm2, or about 500 cells/mm2 to 5,000 cells/mm2. In some cases, the PSCs can be seeded at cell densities of at least about 100 cells/mm2, or at least about 300 cells/mm2, or at least about 700 cells/mm2.
  • A variety of primed pluripotent cell culture medias can be used. Examples include mTESR, MEF conditioned media, StemFit, StemPro, or E8.
  • The culture media used in the apical compartment need not contain BMP. However, the culture media used in the basolateral compartment does contain BMP2, BMP4, or a combination thereof. Depending on pore size of the transwell membranes used, BMP4 can sometimes diffuse to the apical compartment, however this does not affect PGCLC differentiation.
  • The BMP can be used in the basolateral culture media in various amounts. For example, BMP can be included in the basolateral culture media in amounts of at least 0.1 ng/ml, or at least 1 ng/ml, or at about 2 ng/ml or at least 5 ng/ml, or at least 10 ng/ml, or at least 20 ng/ml, or at least 25 ng/ml, or at least 30 ng/ml, or at least 35 ng/ml, or at least 40 ng/ml, or at least 50 ng/ml. In general, the BMP is used in the culture media in amounts less than 200 ng/ml, or less than 150 ng/ml, or less than 100 ng/ml, or less than 75 ng/ml, or less than 60 ng/ml.
  • The time for conversion of starting PSCs into primordial germ cells in the BMP-containing media can vary. For example, the starting cells can be incubated in vessels that provide basolateral BMP for at least about 1 day, or for at least about 2 days, or for at least about 3 days, or for at least about 4 days, or for at least about 5 days, or for at least about 6 days, or for at least about 7 days, or for at least about 8 days, or for at least about 9 days, or for at least about 10 days, or for at least about 11 days, or for at least about 12 days, or for at least about 13 days, or for at least about 14 days.
  • Use of BMP in contact with the basolateral sides of cells modifies epithelial structures those cells to thereby facilitate their differentiation into primordial germ cells.
  • Human Induced Pluripotent Stem Cells (hiPSCs)
  • As described herein a variety of different sources or types of pluripotent stem cells can be used to generate primordial stem cells. However, in some cases induced pluripotent stem cells (iPSCs) can be used.
  • Cells for that are used generating iPSCs are collected from a subject and referred to herein as “starting cells.” A selected starting population of cells may be derived from essentially any source and may be heterogeneous or homogeneous. The term “selected cell” or “selected cells” is also used to refer to starting cells. In certain embodiments, the selected starting cells to be treated as described herein are adult cells, including essentially any accessible adult cell type(s). In other embodiments, the selected starting cells treated according to the invention are adult stem cells, progenitor cells, or somatic cells. In some embodiments, the starting population of cells does not include pluripotent stem cells. In other embodiments, the starting population of cells can include pluripotent stem cells. Accordingly, a starting population of cells that is reprogrammed by the compositions and/or methods described herein, can be essentially any live cell type, particularly a somatic cell type.
  • The starting cells can be treated for a time and under conditions sufficient to convert the starting cells across lineage and/or differentiation boundaries to form induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells are reprogrammed mature cells that have the capacity to differentiate into different mature cell type.
  • The starting cells can be induced to form pluripotent stem cells using either genetic or chemical induction methods. Examples of methods for generating human induced pluripotent stem cells include those described by U.S. Pat. No. 8,058,065 (Yamanaka et al.), WO/2019/165988 by Pei et al., and U.S. Patent Application No. 20190282624 by Deng et al. Induced PSC can also be generated through chemical reprogramming, via JNK pathway inhibition as illustrated by Guan et al. (Nature 605:325-331 (2022)).
  • The iPSCs so obtained can be incubated in any convenient primed pluripotent media. Examples of culture media that can be used include mTESR, MEF conditioned media, StemFit, StemPro, E8, and others.
  • A ROCK inhibitor can be used in the iPSC culture medium, especially prior to incubation with BMP. The ROCK inhibitor can be Y-27632, which is a cell-permeable, highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Y-27632 inhibits both ROCK1 (Ki=220 nM) and ROCK2 (Ki=300 nM). A structure for Y-27632 is shown below.
  • Figure US20250263654A1-20250821-C00001
  • Use of Y-27632 can improve survival of stem cells when they are dissociated to single cells and after thawing the stem cells. Y-27632 can also reduce or block apoptosis of stem cells.
  • The ROCK inhibitor can be used in the culture media in amounts of at least 0.5 uM, or at least 1.0 uM, or at least 2.0 uM, or at least 3.0 uM, or at least 4.0 uM, or at least 5.0 uM, or at least 6.0 uM, or at least 7.0 uM, or at least 8.0 uM, or at least 9.0 uM, or at about 10 uM. In general, the ROCK inhibitor is used in the culture media in amounts less than 30 uM, or less than 25 uM, or less than 20 uM, or less than 15 uM.
  • The ROCK inhibitor can be used in the culture media when the hiPSCs are initially seeded into the vessel (e.g., wells) where the primordial germ cells will be generated. However, the ROCK inhibitor can be removed when the culture media is replaced with media containing BMP.
    • Inhibiting Tight Junction Proteins
  • Epithelial structures are maintained by tight junctions, via key tight junction scaffolding proteins, such as the Zonula-occludens (ZO) family of proteins. Tight junctions form dual-purpose adhesion plaques that endow an epithelium with both barrier and partitioning functions (polarity/directionality) (see FIG. 4 ). Disruption of epithelial tissue structure and apical/basolateral polarity specifically, as illustrated herein, is a key method for generating primordial germ cells.
  • In some cases, tight junction proteins in the PSCs can be inhibited or modified (knocked down or knocked out) to facilitate generation of primordial germ cells. For example, the PSCs or incipient mesoderm-like cells (iMeLCs) can first be genetically modified or pre-treated with a tight junction inhibitor and then the cells can be cultured with BMP. As proof of principle, experiments described herein show that treatment of adherent cultures of ZO1/TIP1 knockdown cells with BMP-4 for 48 hours yielded high numbers of PGC like-cells (PGCLCs).
  • Examples of tight junction inhibitors that can be used include PTPN1 (Tyrosine-protein phosphatase non-receptor type 1), acetylaldehyde, genistein, protein phosphatase 2 (PP2), Clostridium perfringens enterotoxins (and their derived mutants), monoclonal antibodies against Claudin-1 (75A, OM-7D3-B3, 3A2, 6F6), monoclonal antibodies against Claudin-6 (IMAB027), Claudin-2 (1A2), monoclonal antibodies against Claudin-5 (R9, R2, 2B12), monoclonal antibodies against Occludin (1-3, 67-2), and combinations thereof.
  • Chelators can also be used as tight junction inhibitors, including calcium chelators. In some cases one or more of the following chelators can be used: chelator is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), dimercaptosuccinic acid, dimercaprol, or a combination thereof.
  • In some cases, tight junction proteins can be knocked down or knocked out before BMP treatment to facilitate generation of primordial germ cells. Examples of tight junction genes or tight junction proteins to be modified, inhibited, knocked down or knocked out include zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, CLDN7. Pluripotent stem cells primarily express ZO1.
  • The following provides information about some tight junction genes and gene products that can be modified to reduce their expression or functioning.
    • Zonula Occludens
  • Silencing of ZO-1 is sufficient to disrupt the epithelial structure of the pluripotent stem cells. Such epithelial structure serves two purposes: (a) to form a barrier that shields cells from the external (apical) signaling milieu and prevent paracellular diffusion of macromolecules, and (b) to sequester apical/basolateral intracellular components to their respective domains. Therefore, disruption leads to (a) increases in accessibility of the external (apical) signaling milieu to the cells/signaling receptors and (b) loss of sequestration of apical/basolateral cellular components.
  • Loss of ZO1 results in increased sensitivity to the morphogen BMP4, leading to more uniform and prolonged activation of the downstream signaling effector pSMAD1/5. As a result of this change in pSMAD1 signaling dynamics, treatment of adherent cultures of ZO1 knockdown (KD) cells with BMP-4 for 48 hours yields high numbers of PGC like-cells (PGCLCs), which is a name for in vitro derived PGCs that are transcriptionally similar to PGCs derived from human embryos.
  • ZO1 loss at the border between the epiblast and the extraembryonic ectoderm (ExE) in mice has been demonstrated to heighten activation of pSMAD1/5 in that location (Zhang et al. Nat. Commun. 2019), correlating to the location of future PGC specification (Irie et al., Reprod. Med. Biol. 2014).
  • The human ZO1 (TJP1) gene is located on chromosome 15 (location 15q13.1; NC_000015.10 (29699367 . . . 29969049, complement; NC_060939.1 (27490136 . . . 27760675, complement). An amino acid sequence for a human zonula occludens-1 (ZO1) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. Q07157.3; UNIPROT accession no. Q07157) and shown below as SEQ ID NO:1.
  • 1 MSARAAAAKS TAMEETAIWE QHTVTLHRAP GFGFGIAISG
    41 GRDNPHFQSG ETSIVISDVL KGGPAEGQLQ ENDRVAMVNG
    81 VSMDNVEHAF AVQQLRKSGK NAKITIRRKK KVQIPVSRPD
    121 PEPVSDNEED SYDEEIHDPR SGRSGVVNRR SEKIWPRDRS
    161 ASRERSLSPR SDRRSVASSQ PAKPTKVTLV KSRKNEEYGL
    201 RLASHIFVKE ISQDSLAARD GNIQEGDVVL KINGTVTENM
    241 SLTDAKTLIE RSKGKLKMVV QRDERATLLN VPDLSDSIHS
    281 ANASERDDIS EIQSLASDHS GRSHDRPPRR SRSRSPDQRS
    321 EPSDHSRHSP QQPSNGSLRS RDEERISKPG AVSTPVKHAD
    361 DHTPKTVEEV TVERNEKQTP SLPEPKPVYA QVGQPDVDLP
    401 VSPSDGVLPN STHEDGILRP SMKLVKFRKG DSVGLRLAGG
    441 NDVGIFVAGV LEDSPAAKEG LEEGDQILRV NNVDFTNIIR
    481 EEAVLFLLDL PKGEEVTILA QKKKDVYRRI VESDVGDSFY
    521 IRTHEEYEKE SPYGLSFNKG EVFRVVDTLY NGKLGSWLAI
    561 RIGKNHKEVE RGIIPNKNRA EQLASVQYTL PKTAGGDRAD
    601 FWRFRGLRSS KRNLRKSRED LSAQPVQTKF PAYERVVLRE
    641 AGFLRPVTIF GPIADVAREK LAREEPDIYQ IAKSEPRDAG
    681 TDQRSSGIIR LHTIKQIIDQ DKHALLDVTP NAVDRLNYAQ
    721 WYPIVVFLNP DSKQGVKTMR MRLCPESRKS ARKLYERSHK
    761 LRKNNHHLFT TTINLNSMND GWYGALKEAI QQQQNQLVWV
    801 SEGKADGATS DDLDLHDDRL SYLSAPGSEY SMYSTDSRHT
    841 SDYEDTDTEG GAYTDQELDE TLNDEVGTPP ESAITRSSEP
    881 VREDSSGMHH ENQTYPPYSP QAQPQPIHRI DSPGFKPASQ
    921 QKAEASSPVP YLSPETNPAS STSAVNHNVN LTNVRLEEPT
    961 PAPSTSYSPQ ADSLRTPSTE AAHIMLRDQE PSLSSHVDPT
    1001 KVYRKDPYPE EMMRQNHVLK QPAVSHPGHR PDKEPNLTYE
    1041 PQLPYVEKQA SRDLEQPTYR YESSSYTDQF SRNYEHRLRY
    1081 EDRVPMYEEQ WSYYDDKQPY PSRPPFDNQH SQDLDSRQHP
    1121 EESSERGYFP RFEEPAPLSY DSRPRYEQAP RASALRHEEQ
    1161 PAPGYDTHGR LRPEAQPHPS AGPKPAESKQ YFEQYSRSYE
    1201 QVPPQGFTSR AGHFEPLHGA AAVPPLIPSS QHKPEALPSN
    1241 TKPLPPPPTQ TEEEEDPAMK PQSVLTRVKM FENKRSASLE
    1281 TKKDVNDTGS FKPPEVASKP SGAPIIGPKP TSQNQFSEHD
    1321 KTLYRIPEPQ KPQLKPPEDI VRSNHYDPEE DEEYYRKQLS
    1361 YFDRRSFENK PPAHIAASHL SEPAKPAHSQ NQSNFSSYSS
    1401 KGKPPEADGV DRSFGEKRYE PIQATPPPPP LPSQYAQPSQ
    1441 PVTSASLHIH SKGAHGEGNS VSLDFQNSLV SKPDPPPSQN
    1448 KPATFRPPNR EDTAQAAFYP QKSFPDKAPV NGTEQTQKTV
    1521 TPAYNRFTPK PYTSSARPFE RKFESPKFNH NLLPSETAHK
    1561 PDLSSKTPTS PKTLVKSHSL AQPPEFDSGV ETFSIHAEKP
    1601 KYQINNISTV PKAIPVSPSA VEEDEDEDGH TVVATARGIF
    1641 NSNGGVLSSI ETGVSIIIPQ GAIPEGVEQE IYFKVCRDNS
    1681 ILPPLDKEKG ETLLSPLVMC GPHGLKFLKP VELRLPHCDP
    1721 KTWQNKCLPG DPNYLVGANC VSVLIDHF

    The TJP1 gene encodes the ZO1 polypeptide with SEQ ID NO: 1. The TIPI gene is on chromosome 15 (location 15q13.1; NC_000015.10 (29699367 . . . 29969049, complement). A nucleotide sequence that encodes the ZO1 polypeptide with SEQ ID NO: 1 is available as European Nucleotide Archive accession no. L14837, provided below as SEQ ID NO:2.
  • 1 TCCGGGTATG GATGTCAATC TTTTGTCTAC AATGTGAATA
    41 CATTTATCCT TCGGGGACCA TCAAGACTTT CAGGAAAGGC
    81 CCCGCCTGTC TCTGCGCGGC CACTTTGCTG GGACAAAGGT
    121 CAACTGAAGA AGTGGGCAGG CCCGAGGCAG GAGAGATGCT
    161 GAGGAGTCCA TGTGCAGGGG AGGGAAAGGG AGAGGCAGTC
    201 AGGGAGAGGA GGAGGAGGTA CCGCCAGAAG GGGATCCTCC
    241 CGCTCCGAAA ACCAGACACC GGGTCTTGCC CTGTGGTCCA
    281 GGCAGGAGTG CAGTGGTGCA ACCTCAGCTC ACTGCAGCCT
    321 TGACCTCCCC GGGCTCAAGC GATCCTCCGG CCACAGCACT
    361 TGGCTGTTCA GCGGCTGGAG GAGCAGGGCC CCAGGTCCTC
    401 CCCACCCTCA CCTGCTGCTC CCAGGTCGTG GCCGTCTTGC
    441 TCTTCCAGGT CCTTCTCTAG GGATGCAATA TTCACATTGC
    481 TAAGATGCAG GTCTAACGCA GAACCTGTCA ACAGAGCCCC
    521 CCATCATCCA CAGCCCACCC AGCGCTGCAG AGCTCAGGAA
    561 GCCTAGCTGA GGAGGACGAC CGTCCCACCT GGGCTTAGAG
    601 TGAGACCAAG GGCAGAAGGC GTGGGAGTTG CTGGGGCAGC
    641 CAGGGAAGGA CACCCCCAGC CCGTCCTCGC AGCCCCCCAC
    681 AGGCAGTGGG AGGCTTGGCT GTTCCTCCGG CAAAACGGGC
    721 ATGCTCAGTG GGCCGGGCCG GCAGGTTTGC GTGGCCGCTG
    761 AGTTGCCGGC GCCGGCTGAG CCAGCGGACG CCGCGTTCCT
    801 TGGCGGCCGC CGGTTCCCGG GAAGTTACGT GGCGAAGCCG
    841 GCTTCCGAGG AGACGCCGGG AGGCCACGGG TGCTGCTGAC
    881 GGGCGGGCGA CCGGGCGAGG CCGACGTGGC CGGGCTGCGA
    921 AAGCTGCGGG AGGCCGAGTG GGTGACCGCG CTCGGAGGGA
    961 GGTGCCGGTC GGGCGCGCCC CGTGGAGAAG ACCCGGGGGG
    1001 GGCGGGCGCT TCCCGGACTT TTGTCCGAGT TGAATTCCCT
    1041 CCCCCTGGGC CGGGCCCTTC CGTCCGCCCC CGCCCGTGCC
    1081 CCGCTCGCTC TCGGGAGATG TTTATTTGGG CTGTGGCGTG
    1121 AGGAGCGGGG GGGCCAGCGC CGCGGAGTTT CGGGTCCGAG
    1161 GAGCCTCGCG CGGCGCTGGA GAGAGACAAG ATGTCCGCCA
    1201 GAGCTGCGGC CGCCAAGAGC ACAGCAATGG AGGAAACAGC
    1241 TATATGGGAA CAACATACAG TGACGCTTCA CAGGGCTCCT
    1281 GGATTTGGAT TTGGAATTGC AATATCTGGT GGACGAGATA
    1321 ATCCTCATTT TCAGAGTGGG GAAACGTCAA TAGTGATTTC
    1361 AGATGTGCTG AAAGGAGGAC CAGCTGAAGG ACAGCTACAG
    1401 GAAAATGACC GAGTTGCAAT GGTTAACGGA GTTTCAATGG
    1441 ATAATGTTGA ACATGCTTTT GCTGTTCAGC AACTAAGGAA
    1481 AAGTGGGAAA AATGCAAAAA TTACAATTAG AAGGAAGAAG
    1521 AAAGTTCAAA TACCAGTAAG TCGTCCTGAT CCTGAACCAG
    1561 TATCTGATAA TGAAGAAGAT AGTTATGATG AGGAAATACA
    1601 TGATCCAAGA AGTGGCCGGA GTGGTGTGGT TAACAGAAGG
    1641 AGTGAGAAGA TTTGGCCGAG GGATAGAAGT GCAAGTAGAG
    1681 AGAGGAGCTT GTCCCCGCGG TCAGACAGGC GGTCAGTGGC
    1721 TTCCAGCCAG CCTGCTAAAC CTACTAAAGT CACACTGGTG
    1761 AAATCCCGGA AAAATGAAGA ATATGGTCTT CGATTGGCAA
    1801 GCCATATATT TGTTAAGGAA ATTTCACAAG ATAGTTTGGC
    1841 AGCAAGAGAT GGCAATATTC AAGAAGGTGA TGTTGTATTG
    1881 AAGATAAATG GTACTGTGAC AGAAAATATG TCATTGACAG
    1921 ATGCAAAGAC ATTGATAGAA AGGTCTAAAG GCAAATTAAA
    1961 AATGGTAGTT CAAAGAGATG AACGGGCTAC GCTATTGAAT
    2001 GTCCCTGATC TTTCTGACAG CATCCACTCT GCTAATGCCT
    2041 CTGAGAGAGA CGACATTTCA GAAATTCAGT CACTGGCATC
    2081 AGATCATTCT GGTCGATCAC ACGATAGGCC TCCCCGCCGC
    2121 AGCCGGTCAC GATCTCCTGA CCAGCGGTCA GAGCCTTCTG
    2161 ATCATTCCAG GCACTCGCCG CAGCAGCCAA GCAATGGCAG
    2201 TCTCCGGAGT AGAGATGAAG AGAGAATTTC TAAACCTGGG
    2241 GCTGTCTCAA CTCCTGTAAA GCATGCTGAT GATCACACAC
    2281 CTAAAACAGT GGAAGAAGTT ACAGTTGAAA GAAATGAGAA
    2321 ACAAACACCT TCTCTTCCAG AACCAAAGCC TGTGTATGCC
    2361 CAAGTTGGCA ACCAGATGTG GATTTACCTG TCAGTCCATC
    2401 TGATGGTGTC CTACCTAATT CAACTCATGA AGATGGGATT
    2441 TCTTCGGCCC AGCATGAAAT TGGTAAAATT CAGAAAAGGA
    2481 GATAGTGTGG GTTTGCGGCT GGCTGGTGGA AATGATGTTG
    2521 GAATATTTGT AGCTGGCGTT CTAGAAGATA GCCCTGCAGC
    2561 CAAGGAAGGC TTAGAGGAAG GTGATCAAAT TCTCAGGGTA
    2601 AACAACGTAG ATTTTACAAA TATCATAAGA GAAGAAGCCG
    2641 TCCTTTTCCT GCTTGACCTC CCTAAAGGAG AAGAAGTGAC
    2681 CATATTGGCT CAGAAGAAGA AGGATGTTTA TCGTCGCATT
    2721 GTAGAATCAG ATGTAGGAGA TTCTTTCTAT ATTAGAACCC
    2761 ATTTTGAATA TGAAAAGGAA TCTCCCTATG GACTTAGTTT
    2801 TAACAAAGGA GAGGTGTTCC GTGCTGTGGA TACCTTGTAC
    2841 AATGGAAAAC TGGGCTCTTG GCTTGCTATT CGAATTGGTA
    2881 AAAATCATAA GGAGGTAGAA CGAGGCATCA TCCCTAATAA
    2921 GAACAGAGCT GAGCAGCTAG CCAGTGTACA GTATACACTT
    2961 CCAAAAACAG CAGGCGGAGA CCGTGCTGAC TTCTGGAGAT
    3001 TCAGAGGTCT TCGCAGCTCC AAGAGAAATC TTCGAAAAAG
    3041 CAGAGAGGAT TTGTCCGCTC AGCCTGTTCA AACAAAGTTT
    3081 CCAGCTTATG AAAGAGTGGT TCTTCGAGAA GCTGGATTTC
    3121 TGAGGCCTGT AACCATTTTT GGACCAATAG CTGATGTTGC
    3161 CAGAGAAAAG CTGGCAAGAG AAGAACCAGA TATTTATCAA
    3201 ATTGCAAAGA GTGAACCACG AGACGCTGGA ACTGACCAAC
    3241 GTAGCTCTGG CTATATTCGC CTGCATACAA TAAAGCAAAT
    3281 CATAGATCAA GACAAACATG CTTTATTAGA TGTAACACCA
    3321 AATGCAGTTG ATCGTCTTAA CTATGCCCAG TGGTATCCAA
    3361 TTGTTGTATT TCTTAACCCT GATTCTAAGC AAGGAGTAAA
    3401 AACAATGAGA ATGAGGTTAT GTCCAGAATC TCGGAAAAGT
    3441 GCCAGGAAGT TATACGAGCG ATCTCATAAA CTTGCTAAAA
    3481 ATAATCACCA TCTTTTTACA ACTACAATTA ACTTAAATTC
    3521 AATGAATGAT GGTTGGTATG GTGCGCTGAA AGAAGCAGTT
    3561 CAACAACAGC AAAACCAGCT GGTATGGGTT TCCGAGGGAA
    3601 AGGCGGATGG TGCTACAAGT GATGACCTTG ATTTGCATGA
    3641 TGATCGTCTG TCCTACCTGT CAGCTCCAGG TAGTGAATAC
    3681 TCAATGTATA GCACGGACAG TAGACACACT TCTGACTATG
    3721 AAGACACAGA CACAGAAGGC GGGGCCTACA CTGATCAAGA
    3761 ACTAGATGAA ACTCTTAATG ATGAGGTTGG GACTCCACCG
    3801 GAGTCTGCCA TTACACGGTC CTCTGAGCCT GTAAGAGAGG
    3841 ACTCCTCTGG AATGCATCAT GAAAACCAAA CATATCCTCC
    3881 TTACTCACCA CAAGCGCAGC CACAACCAAT TCATAGAATA
    3921 GACTCCCCTG GATTTAAGCC AGCCTCTCAA CAGAAAGCAG
    3961 AAGCTTCATC TCCAGTCCCT TACCTTTCGC CTGAAACAAA
    4001 CCCAGCATCA TCAACCTCTG CTGTTAATCA TAATGTAAAT
    4041 TTAACTAATG TCAGACTGGA GGAGCCCACC CCAGCTCCTT
    4081 CCACCTCTTA CTCACCACAA GCTGATTCTT TAAGAACACC
    4121 AAGTACTGAG GCAGCTCACA TAATGCTAAG AGATCAAGAA
    4161 CCATCATTGT CGTCGCATGT AGATCCAACA AAGGTGTATA
    4201 GAAAGGATCC ATATCCCGAG GAAATGATGA GGCAGAACCA
    4241 TGTTTTGAAA CAGCCAGCCG TTAGTCACCC AGGGCACAGG
    4281 CCAGACAAAG AGCCTAATCT GACCTATGAA CCCCAACTCC
    4321 CATACGTAGA GAAACAAGCC AGCAGAGACC TCGAGCAGCC
    4361 CACATACAGA TACGAGTCCT CAAGCTATAC GGACCAGTTT
    4401 TCTCGAAACT ATGAACATCG TCTGCGATAC GAAGATCGCG
    4441 TCCCCATGTA TGAAGAACAG TGGTCATATT ATGATGACAA
    4481 ACAGCCCTAC CCATCTCGGC CACCTTTTGA TAATCAGCAC
    4521 TCTCAAGACC TTGACTCCAG ACAGCATCCC GAAGAGTCCT
    4561 CAGAACGAGG GTACTTTCCA CGTTTTGAAG AGCCAGCCCC
    4601 TCTGTCTTAC GACAGCAGAC CACGTTACGA ACAGGCACCT
    4641 AGAGCATCCG CCCTGCGGCA CGAAGAGCAG CCAGCTCCTG
    4681 GGTATGACAC ACATGGTAGA CTCAGACCGG AAGCCCAGCC
    4721 CCACCCTTCA GCAGGGCCCA AGCCTGCAGA GTCCAAGCAG
    4761 TATTTTGAGC AATATTCACG CAGTTACGAG CAAGTACCAC
    4801 CCCAAGGATT TACCTCTAGA GCAGGTCATT TTGAGCCTCT
    4841 CCATGGTGCT GCAGCTGTCC CTCCGCTGAT ACCTTCATCT
    4881 CAGCATAAGC CAGAAGCTCT GCCTTCAAAC ACCAAACCAC
    4921 TGCCTCCACC CCCAACTCAA ACCGAAGAAG AGGAAGATCC
    4961 AGCAATGAAG CCACAGTCTG TACTCACCAG AGTTAAGATG
    5001 TTTGAAAACA AAAGATCTGC ATCCTTAGAG ACCAAGAAGG
    5041 ATGTAAATGA CACTGGCAGT TTTAAGCCTC CAGAAGTAGC
    5081 ATCTAAACCT TCAGGTGCTC CCATCATTGG TCCCAAACCC
    5121 ACTTCTCAGA ATCAATTCAG TGAACATGAC AAAACTCTGT
    5161 ACAGGATCCC AGAACCTCAA AAACCTCAAC TGAAGCCACC
    5201 TGAAGATATT GTTCGGTCCA ATCATTATGA CCCTGAAGAA
    5241 GATGAAGAAT ATTATCGAAA ACAGCTGTCA TACTTTGACC
    5281 GAAGAAGTTT TGAGAATAAG CCTCCTGCAC ACATTGCTGC
    5321 CAGCCATCTC TCCGAGCCTG CAAAGCCAGC TCATTCTCAG
    5361 AATCAATCAA ATTTTTCTAG TTATTCTTCA AAGGGAAAGC
    5401 CTCCTGAAGC TGATGGTGTG GATAGATCAT TTGGCGAGAA
    5441 ACGCTATGAA CCCATCCAGG CCACTCCCCC TCCTCCTCCA
    5481 TTGCCCTCGC AGTATGCCCA GCCATCTCAG CCTGTCACCA
    5521 GCGCGTCTCT CCACATACAT TCTAAGGGAG CACATGGTGA
    5561 AGGTAATTCA GTGTCATTGG ATTTTCAGAA TTCCTTAGTG
    5601 TCCAAACCAG ACCCACCTCC ATCTCAGAAT AAGCCAGCAA
    5641 CTTTCAGACC ACCAAACCGA GAAGATACTG CTCAGGCAGC
    5681 TTTCTATCCC CAGAAAAGTT TTCCAGATAA AGCCCCAGTT
    5721 AATGGAACTG AACAGACTCA GAAAACAGTC ACTCCAGCAT
    5761 ACAATCGATT CACACCAAAA CCATATACAA GTTCTGCCCG
    5801 ACCATTTGAA CGCAAGTTTG AAAGTCCTAA ATTCAATCAC
    5841 AATCTTCTGC CAAGTGAAAC TGCACATAAA CCTGACTTGT
    5881 CTTCAAAAAC TCCCACTTCT CCAAAAACTC TTGTGAAATC
    5921 GCACAGTTTG GCACAGCCTC CTGAGTTTGA CAGTGGAGTT
    5961 GAAACTTTCT CTATCCATGC AGAGAAGCCT AAATATCAAA
    6001 TAAATAATAT CAGCACAGTG CCTAAAGCTA TTCCTGTGAG
    6041 TCCTTCAGCT GTGGAAGAGG ATGAAGATGA AGATGGTCAT
    6081 ACTGTGGTGG CCACAGCCCG AGGCATATTT AACAGCAATG
    6121 GGGGCGTGCT GAGTTCCATA GAAACTGGTG TTAGTATAAT
    6161 TATCCCTCAA GGAGCCATTC CCGAAGGAGT TGAGCAGGAA
    6201 ATCTATTTCA AGGTCTGCCG GGACAACAGC ATCCTTCCAC
    6241 CTTTAGATAA AGAGAAAGGT GAAACACTGC TGAGTCCTTT
    6281 GGTGATGTGT GGTCCCCATG GCCTCAAGTT CCTGAAGCCT
    6321 GTGGAGCTGC GCTTACCACA CTGTGATCCT AAAACCTGGC
    6361 AAAACAAGTG TCTTCCCGGA GATCCAAATT ATCTCGTTGG
    6401 AGCAAACTGT GTTTCTGTCC TTATTGACCA CTTTTAACTC
    6441 TTGAAATATA GGAACTTAAA TAATGTGAAA CTGGATTAAA
    6481 CTTAATCTAA ATGGAACCAC TCTATCAAGT ATTATACCTT
    6521 TTTTAGAGTT GATACTACAG TTTGTTAGTA TGAGGCATTT
    6561 GTTTGAACTG ATAAAGATGA GTGAGCATGC CCCTGAACCA
    6601 TGGTCGGAAA ACATGCTACA CACTGCATGT TTGTGATTGA
    6641 CGGGACTGTT GGTATTGGCT AGAGGTTCAA AGATATTTTG
    6681 CTTTGTGATT TTTGTAATTT TTTTATCGTC ACTGCTTAAC
    6721 TTCACATATT GATTTCCGTT AAAATACCAG CCAGTAAATG
    6761 GGGGTGCATT TGAGGTCTGT TCTTTCCAAA GTACACTGTT
    6801 TCAAACTTTA CTATGGCCCT GGCCTAGCAT ACGTACACAT
    6841 TTTATTTTAT TATGCATGAA GTAATATGCA CACATTTTTT
    6881 AAATGCACCT GGAATATATA ACCAGTGTTG TGGATTTAAC
    6921 AGAAATGTAC AGCAAGGAGA TTTACAACTG GGGGAGGGTG
    6961 AAGTGAAGAC AATGACTTAC TGTACATGAA AACACATTTT
    7001 TCTTAGGGAA GGATACAAAA GCATGTGAGA CTGGTTCCAT
    7041 GGCCTCTTCA GATCTCTAAC TTCACCATAT TACCACAGAC
    7081 ATACTAACCA GCAGAAATGC CTTACCCTCA TGTTCTTAAT
    7121 TCTTAGCTCA TTCTCCTTGT GTTACTAAGT TTTTATGGCT
    7161 TTTGTGCATT ATCTAGATAC TGTATCATGA CAAAGACTGA
    7201 GTACGTTGTG CATTTGGTGG TTTCAGAAAT GTGTTATCAC
    7241 CCAGAAGAAA ATAGTGGTGT GATTTGGGGA TATTTTTTTC
    7281 TTTTCTTTTC TTTTCTTTTT TTTTTTTTTT TGACAAGGGG
    7321 CAGTGGTGGT TTTCTGTTCT TTCTGGCTAT GCATTTGAAA
    7361 ATTTTGATGT TTTAAGGATG CTTGTACATA ATGCGTGCAT
    7401 ACCACTTTTG TTCTTGGTTT GTAAATTAAC TTTTATAAAC
    7441 TTTACCTTTT TTATACATAA ACAAGACCAC GTTTCTAAAG
    7481 GCTACCTTTG TATTCTCTCC TGTACCTCTT GAGCCTTGAA
    7521 CTTTGACCTC TGCAGCAATA AAGCAGCGTT TCTATGACAC
    7561 ATGCAAGGTC ATTTTTTTTA AGAAAAAGGA TGCACAGAGT
    7601 TGTTACATTT TTAAGTGCTG CATTTAAAAG ATACAGTTAC
    7641 TCAGAATTCT CTAGTTTGAT TAAATTCTTG CAAAGTATCC
    7681 CTACTGTAAT TTGTGATACA ATGCTGTGCC CTAAAGTGTA
    7721 TTTTTTTACT AATAGACAAT TTATTATGAC ACATCAGCAC
    7761 GATTTCTGTT TAAATAATAC ACCACTACAT TCTGTTAATC
    7800 ATTAGGTGTG ACTGAATTTC TTTTGCCGTT ATTAAAAATC
    7841 TCAAATTTCT AAATCTCCAA AATAAAACTT TTTAAAATAA
    7881 AAAAAAAT
  • An amino acid sequence for a human zonula occludens-2 (Z (2) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. Q9UDY2.2; UNIPROT accession no. Q9UDY2) and shown below as SEQ ID NO:3.
  • 1 MPVRGDRGFP PRRELSGWLR APGMEELIWE QYTVTLQKDS
    41 KRGFGIAVSG GRDNPHFENG ETSIVISDVL PGGPADGLLQ
    81 ENDRVVMVNG TPMEDVLHSF AVQQLRKSGK VAAIVVKRPR
    121 KVQVAALQAS PPLDQDDRAF EVMDEFDGRS FRSGYSERSR
    161 LNSHGGRSRS WEDSPERGRP HERARSRERD LSRDRSRGRS
    201 LERGLDQDHA RTRDRSRGRS LERGLDHDFG PSRDRDRDRS
    241 RGRSIDQDYE RAYHRAYDPD YERAYSPEYR RGARHDARSR
    281 GPRSRSREHP HSRSPSPEPR GRPGPIGVLL MKSRANEEYG
    321 LRLGSQIFVK EMTRTGLATK DGNLHEGDII LKINGTVTEN
    361 MSLTDARKLI EKSRGKLQLV VLRDSQQTLI NIPSLNDSDS
    401 EIEDISEIES NRSFSPEERR HQYSDYDYHS SSEKLKERPS
    441 SREDTPSRLS RMGATPTPEK STGDIAGTVV PETNKEPRYQ
    481 EDPPAPQPKA APRTFLRPSP EDEAIYGPNT KMVRFKKGDS
    521 VGLRLAGGND VGIFVAGIQE GTSAEQEGLQ EGDQILKVNT
    561 QDFRGLVRED AVLYLLEIPK GEMVTILAQS RADVYRDILA
    601 CGRGDSFFIR SHFECEKETP QSLAFTRGEV FRVVDTLYDG
    641 KLGNWLAVRI GNELEKGLIP NKSRAEQMAS VQNAQRDNAG
    681 DRADFWRMRG QRSGVKKNLR KSREDLTAVV SVSTKFPAYE
    721 RVLLREAGFK RPVVLEGPIA DIAMEKLANE LPDWFQTAKT
    761 EPKDAGSEKS TGVVRLNTVR QIIEQDKHAL LDVTPKAVDL
    801 LNYTQWFPIV IFFNPDSRQG VKTMRQRLNP TSNKSSRKLF
    841 DQANKLKKTC AHLFTATINL NSANDSWFGS LKDTIQHQQG
    881 EAVWVSEGKM EGMDDDPEDR MSYLTAMGAD YLSCDSRLIS
    921 DFEDTDGEGG AYTDNELDEP AEEPLVSSIT RSSEPVQHEE
    961 SIRKPSPEPR AQMRRAASSD QLRDNSPPPA FKPEPPKAKT
    1001 QNKEESYDFS KSYEYKSNPS AVAGNETPGA STKGYPPPVA
    1041 AKPTFGRSIL KPSTPIPPQE GEEVGESSEE QDNAPKSVLG
    1081 KVKIFEKMDH KARLQRMQEL QEAQNARIEI AQKHPDIYAV
    1121 PIKTHKPDPG TPQHTSSRPP EPQKAPSRPY QDTRGSYGSD
    1161 AEEEEYRQQL SEHSKRGYYG QSARYRDTEL

    The TJP2 gene encodes the ZO2 polypeptide with SEQ ID NO:3. The TJP2 gene is on chromosome 9 (location NC_000009.12 (69121006 . . . 69255208)). A nucleotide sequence that encodes the ZO2 polypeptide with SEQ ID NO:3 is available as European Nucleotide Archive accession no. L27476, provided below as SEQ ID NO: 4.
  • 1 TGCCCAGGAG GAGTAGGAGC AGGAGCAGAA GCAGAAGCGG
    41 GGTCCGGAGC TGCGCGCCTA CGCGGGACCT GTGTCCGAAA
    81 TGCCGGTGCG AGGAGACCGC GGGTTTCCAC CCCGGCGGGA
    121 GCTGTCAGGT TGGCTCCGCG CCCCAGGCAT GGAAGAGCTG
    161 ATATGGGAAC AGTACACTGT GACCCTACAA AAGGATTCCA
    201 AAAGAGGATT TGGAATTGCA GTGTCCGGAG GCAGAGACAA
    241 CCCCCACTTT GAAAATGGAG AAACGTCAAT TGTCATTTCT
    281 GATGTGCTCC CGGGTGGGCC TGCTGATGGG CTGCTCCAAG
    321 AAAATGACAG AGTGGTCATG GTCAATGGCA CCCCCATGGA
    361 GGATGTGCTT CATTCGTTTG CAGTTCAGCA GCTCAGAAAA
    401 AGTGGGAAGG TCGCTGCTAT TGTGGTCAAG AGGCCCCGGA
    441 AGGTCCAGGT GGCCGCACTT CAGGCCAGCC CTCCCCTGGA
    481 TCAGGATGAC CGGGCTTTTG AGGTGATGGA CGAGTTTGAT
    521 GGCAGAAGTT TCCGGAGTGG CTACAGCGAG AGGAGCCGGC
    561 TGAACAGCCA TGGGGGGCGC AGCCGCAGCT GGGAGGACAG
    601 CCCGGAAAGG GGGCGTCCCC ATGAGCGGGC CCGGAGCCGG
    641 GAGCGGGACC TCAGCCGGGA CCGGAGCCGT GGCCGGAGCC
    681 TGGAGCGGGG CCTGGACCAA GACCATGCGC GCACCCGAGA
    721 CCGCAGCCGT GGCCGGAGCC TGGAGCGGGG CCTGGACCAC
    761 GACTTTGGGC CATCCCGGGA CCGGGACCGT GACCGCAGCC
    801 GCGGCCGGAG CATTGACCAG GACTACGAGC GAGCCTATCA
    841 CCGGGCCTAC GACCCAGACT ACGAGCGGGC CTACAGCCCG
    881 GAGTACAGGC GCGGGGCCCG CCACGATGCC CGCTCTCGGG
    921 GACCCCGAAG CCGCAGCCGC GAGCACCCGC ACTCACGGAG
    961 CCCCAGCCCC GAGCCTAGGG GGCGGCCGGG GCCCATCGGG
    1001 GTCCTCCTGA TGAAAAGCAG AGCGAACGAA GAGTATGGTC
    1041 TCCGGCTTGG GAGTCAGATC TTCGTAAAGG AAATGACCCG
    1081 AACGGGTCTG GCAACTAAAG ATGGCAACCT TCACGAAGGA
    1121 GACATAATTC TCAAGATCAA TGGGACTGTA ACTGAGAACA
    1161 TGTCTTTAAC GGATGCTCGA AAATTGATAG AAAAGTCAAG
    1201 AGGAAAACTA CAGCTAGTGG TGTTGAGAGA CAGCCAGCAG
    1241 ACCCTCATCA ACATCCCGTC ATTAAATGAC AGTGACTCAG
    1281 AAATAGAAGA TATTTCAGAA ATAGAGTCAA CCCGATCATT
    1321 TTCTCCAGAG GAGAGACGTC ATCAGTATTC TGATTATGAT
    1361 TATCATTCCT CAAGTGAGAA GCTGAAGGAA AGGCCAAGTT
    1401 CCAGAGAGGA CACGCCGAGC AGATTGTCCA GGATGGGTGC
    1441 GACACCCACT CCCTTTAAGT CCACAGGGGA TATTGCAGGC
    1481 ACAGTTGTCC CAGAGACCAA CAAGGAACCC AGATACCAAG
    1521 AGGAACCCCC AGCTCCTCAA CCAAAAGCAG CCCCGAGAAC
    1561 TTTTCTTCGT CCTAGTCCTG AAGATGAAGC AATATATGGC
    1601 CCTAATACCA AAATGGTAAG GTTCAAGAAG GGAGACAGCG
    1641 TGGGCCTCCG GTTGGCTGGT GGCAATGATG TCGGGATATT
    1681 TGTTGCTGGC ATTCAAGAAG GGACCTCGGC GGAGCAGGAG
    1721 GGCCTTCAAG AAGGAGACCA GATTCTGAAG GTGAACACAC
    1761 AGGATTTCAG AGGATTAGTG CGGGAGGATG CCGTTCTCTA
    1801 CCTGTTAGAA ATCCCTAAAG GTGAAATGGT GACCATTTTA
    1841 GCTCAGAGCC GAGCCGATGT GTATAGAGAC ATCCTGGCTT
    1881 GTGGCAGAGG GGATTCGTTT TTTATAAGAA GCCACTTTGA
    1921 ATGTGAGAAG GAAACTCCAC AGAGCCTGGC CTTCACCAGA
    1961 GGGGAGGTCT TCCGAGTGGT AGACACACTG TATGACGGCA
    2001 AGCTGGGCAA CTGGCTGGCT GTGAGGATTG GGAACGAGTT
    2041 GGAGAAAGGC TTAATCCCCA ACAAGAGCAG AGCTGAACAA
    2081 ATGGCCAGTG TTCAAAATGC CCAGAGAGAC AACGCTGGGG
    2121 ACCGGGCAGA TTTCTGGAGA ATGCGTGGCC AGAGGTCTGG
    2161 GGTGAAGAAG AACCTGAGGA AAAGTCGGGA AGACCTCACA
    2201 GCTGTTGTGT CTGTCAGCAC CAAGTTCCCA GCTTATGAGA
    2241 GGGTTTTGCT GCGAGAAGCT GGTTTCAAGA GACCTGTGGT
    2281 CTTATTCGGC CCCATAGCTG ATATAGCAAT GGAAAAATTG
    2321 GCTAATGAGT TACCTGACTG GTTTCAAACT GCTAAAACGG
    2361 AACCAAAAGA TGCAGGATCT GAGAAATCCA CTGGAGTGGT
    2401 CCGGTTAAAT ACCGTGAGGC AAGTTATTGA ACAGGATAAG
    2441 CATGCACTAC TGGATGTGAC TCCGAAAGCT GTGGACCTGT
    2481 TGAATTACAC CCAGTGGTTC TCAATTGTGA TTTCTTTCAC
    2521 GCCAGACTCC AGACAAGGTG TCAACACCAT GAGACAAAGG
    2561 TTAGACCCAA CGTCCAACAA TAGTTCTCGA AAGTTATTTG
    2601 ATCACGCCAA CAAGCTTAAA AAAACGTGTG CACACCTTTT
    2641 TACAGCTACA ATCAACCTAA ATTCAGCCAA TGATAGCTGG
    2681 TTTGGCAGCT TAAAGGACAC TATTCAGCAT CAGCAAGGAG
    2721 AAGCGGTTTG GGTCTCTGAA GGAAAGATGG AAGGGATGGA
    2761 TGATGACCCC GAAGACCGCA TGTCCTACTT AACTGCCATG
    2801 GGCGCAGACT ATCTGAGTTG CGACAGCCGC CTCATCAGTG
    2841 ACTTTGAAGA CACGGACGGT GAAGGAGGCG CCTACACTGA
    2881 CAATGAGCTG GATGAGCCAG CCGAGGAGCC GCTGGTGTCG
    2921 TCCATCACCC GCTCCTCGGA GCCGGTGCAG CACGAGGAGA
    2961 GCATAAGGAA ACCCAGCCCA GAGCCACGAG CTCAGATGAG
    3001 GAGGGCTGCT AGCAGCGATC AACTTAGGGA CAATAGCCCG
    3041 CCCCCAGCAT TCAAGCCAGA GCCGTCCAAG GCCAAAACCC
    3081 AGAACAAAGA AGAATCCTAT GACTTCTCCA AATCCTATGA
    3121 ATATAAGTCA AACCCCTCTG CCGTTGCTGG TAATGAAACT
    3161 CCTGGGGCAT CTACCAAAGG TTATCCTCCT CCTGTTGCAG
    3201 CAAAACCTAC CTTTGGGGGG TCTATACTGA AGCCCTCCAC
    3241 TCCCATCCCT CCTCAAGAGG GTGAGGAGGT GGGAGAGAGC
    3281 AGTGAGGAGC AAGATAATGC TCCCAAATCA GTCCTGGGCA
    3321 AAGTCAAAAT ATTTGGAGAA GATGGATCAC AAGGGCCAGG
    3361 GTTACAAGAG AATGCAGGAG CTCCAGGAAG CACAGAATGC
    3401 AAGGATCGAA ATTGCCCAGA AGCATCCTGA TATCTATGCA
    3441 GTTCCAATCA AAACGCACAA GCCAGACCCT GGCACGCCCC
    3481 AGCACACGAG TTCCAGACCC CCTGAGCCAC AGAAAGCTCC
    3521 TTCCAGACCT TATCAGGATA CCAGAGGAAG TTATGGCAGT
    3561 GATGCCGAGG AGGAGGAGTA CCGCCAGCAG CTGTCAGAAC
    3601 ACTCCAAGCG CGGTTACTAT GGCCAGTCTG CCCGATACCG
    3641 GGACACAGAA TTATAGATGT CTGAGCACGG ACTCTCCCAG
    3681 GCCTGCCTGC ATGGCATCAG ACTAGCCACT CCTGCCAGGC
    3721 CGCCGGGATG GTTCTTCTCC AGTTAGAATG CACCATGGAG
    3761 ACGTGGTGGG ACTCCAGCTC GTGTGTCCTC ATGGAGAACC
    3801 CAGGGGACAG CTGGTGCAAA TTCAGAACTG AGGGCTCTGT
    3841 TTGTGGGACT GGGTTAGAGG AGTCTGTGGC TTTTTGTTCA
    3881 GAATTAAGCA GAACACTGCA GTCAGATCCT GTTACTTGCT
    3921 TCAGTGGACC GAAATCTGTA TTCTGTTTGC GTACTTGTAA
    3961 TATGTATATT AAGAAGCAAT AACTATTTTT CCTCATTAAT
    4001 AGCTGCCTTC AAGGACTGTT TCAGTGTGAG TCAGAATGTG
    4041 AAAAAGGAAT AAAAAATACT GTTGGGCTCA AACTAAATTC
    4081 AAAGAAGTAC TTTATTGCAA CTCTTTTAAG TGCCTTGGAT
    4121 GAGAAGTGTC TTAAATTTTC TTCCTTTGAA GCTTTAGGCA
    4161 GAGCCATAAT GGACTAAAAC ATTTTGACTA AGTTTTTATA
    4201 CCAGCTTAAT AGCTGTAGTT TTCCCTGCAC TGTGTCATCT
    4241 TTTCAAGGCA TTTGTCTTTG TAATATTTTC CATAAATTTG
    4281 GACTGTCTAT ATCATAACTA TACTTGATAG TTTGGCTATA
    4321 AGTGCTCAAT AGCTTGAAGC CCAAGAAGTT GGTATCGAAA
    4361 TTTGTTGTTT GTTTAAACCC AAGTGCTGCA CAAAAGCAGA
    4401 TACTTGAGGA AAACACTATT TCCAAAAGCA CATGTATTGA
    4441 CAACAGTTTT ATAATTTAAT AAAAAGGAAT ACATTGCAAT
    4481 CCGT
  • An amino acid sequence for a human zonula occludens-3 (ZO3) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. EAW69293.1; UNIPROT accession no. 095049) and shown below as SEQ ID NO:5.
  • 1 MEELTIWEQH TATLSKDPRR GFGIAISGGR DRPGGSMVVS
    41 DVVPGGPAEG RLQTGDHIVM VNGVSMENAT SAFAIQILKT
    81 CTKMANITVK RPRRIHLPAT KASPSSPGRQ DSDEDDGPQR
    121 VEEVDQGRGY DGDSSSGSGR SWDERSRRPR PGRRGRAGSH
    161 GRRSPGGGSE ANGLALVSGF KRLPRQDVQM KPVKSVLVKR
    201 RDSEEFGVKL GSQIFIKHIT DSGLAARHRG LQEGDLILQI
    241 NGVSSQNLSL NDTRRLIEKS EGKLSLLVLR DRGQFLVNIP
    281 PAVSDSDSSP LEEGVTMADE MSSPPADISD LASELSQAPP
    321 SHIPPPPRHA QRSPEASQTD SPVESPRLRR ESSVDSRTIS
    361 EPDEQRSELP RESSYDIYRV PSSQSMEDRG YSPDTRVVRF
    401 LKGKSIGLRL AGGNDVGIFV SGVQAGSPAD GQGIQEGDQI
    441 LQVNDVPFQN LTREEAVQFL LGLPPGEEME LVTQRKQDIF
    481 WKMVQSRVGD SFYIRTHFEL EPSPPSGLGF TRGDVFHVLD
    521 TLHPGPGQSH ARGGHWLAVR MGRDLREQER GIIPNQSRAE
    561 QLASLEAAQR AVGVGPGSSA GSNARAEFWR LRGLRRGAKK
    601 TTQRSREDLS ALTRQGRYPP YERVVLREAS FKRPVVILGP
    641 VADIAMQKLT AEMPDQFEIA ETVSRTDSPS KIIKLDTVRV
    681 IAEKDKHALL DVTPSAIERL NYVQYYPIVV FFIPESRPAL
    721 KALRQWLAPA SRRSTRRLYA QAQKLRKHSS HLFTATIPLN
    761 GTSDTWYQEL KAIIREQQTR PIWTAEDQLD GSLEDNLDLP
    801 HHGLADSSAD LSCDSRVNSD YETDGEGGAY TDGEGYTDGE
    841 GGPYTDVDDE PPAPALARSS EPVQADESQS PRDRGRISAH
    881 QGAQVDSRHP QGQWRQDSMR TYEREALKKK FMRVHDAESS
    921 DEDGYDWGPA TDL

    The TJP3 gene encodes the ZO3 polypeptide with SEQ ID NO:5. The TJP3 gene is on chromosome 19 (location NC_000019.10 (3708384 . . . 3750813)). A nucleotide sequence that encodes the ZO3 polypeptide with SEQ ID NO:5 is available as European Nucleotide Archive accession no. AK091118, provided below as SEQ ID NO: 6.
  • 1 AGTTCCACTG GCAGGCGACC TGCCTCCCTG TTGCCACCAC
    41 AAGAGAGGAA AAGTTGGTCA AACAGGTGGG GAGGCCAGAG
    61 CTACAAGCCT CGGGTTCCCT CCCCACCACC CGTGCCAGGC
    121 AGGCACCCGG GCCCTGGCAC CTGCTGCCTG CCCAGAGGCC
    161 ACCCAGCCTC CTAGACAGGT GGCTGACATG GAGGAGCTGA
    201 CCATCTGGGA ACAGCACACG GCCACACTGT CCAAGGACCC
    241 CCGCCGGGGC TTTGGCATTG CGATCTCTGG AGGCCGAGAC
    281 CGGCCCGGTG GATCCATGGT TGTATCTGAC GTGGTACCTG
    321 GAGGGCCGGC GGAGGGCAGG CTACAGACAG GCGACCACAT
    361 TGTCATGGTG AACGGGGTTT CCATGGAGAA TGCCACCTCC
    401 GCGTTTGCCA TTCAGATACT CAAGACCTGC ACCAAGATGG
    441 CCAACATCAC AGTGAAACGT CCCCGGAGGA TCCTCCTGCC
    481 CGCCACCAAA GCCAGCCCCT CCAGCCCAGG GCGCCAGGAC
    521 TCGGATGAAG ACGATGGGCC CCAGCGGGTG GAGGAGGTGG
    561 ACCAGGGCCG GGGCTATGAC GGCGACTCAT CCAGTGGCTC
    601 CGGCCGCTCC TGGGACGAGC GCTCCCGCCG GCCGAGGCCT
    641 GGTCGCCGGG GCCGGGCCGG CAGCCATGGG CGTAGGAGCC
    681 CAGGTGGTGG CTCTGAGGCC AACGGGCTGG CCCTGGTGTC
    721 CGGCTTTAAG CGGCTGCCAC GGCAGGACGT GCAGATGAAG
    761 CCTGTGAAGT CAGTGCTGGT GAAGAGGAGA GACAGCGAAG
    801 AGTTTGGCGT CAAGCTGGGC AGTCAGATCT TCATCAAGCA
    841 CATTACAGAT TCGGGCCTGG CTGCCCGGCA CCGTGGGCTG
    881 CAGGAAGGAG ATCTCATTCT ACAGATCAAC GGGGTGTCTA
    921 GCCAGAACCT GTCACTGAAC GACACCCGGC GACTGATTGA
    961 GAAGTCAGAA GGGAAGCTAA GCCTGCTGGT GCTGAGAGAT
    1001 CGTGGGCAGT TCCTGGTGAA CATTCCGCCT GCTGTCAGTG
    1041 ACAGCGACAG CTCGCCATTG GAGGACATCT CGGACCTCGC
    1081 CTCGGAGCTA TCGCAGGCAC CACCATCCCA CATCCCACCA
    1121 CCACCCCGGC ATGCTCAGCG GAGCCCCGAG GCCAGCCAGA
    1161 CCGACTCTCC CGTGGAGAGT CCCCGGCTTC GGCGGGAAAG
    1201 TTCAGTAGAT TCCAGAACCA TCTCGGAACC AGATGAGCAA
    1241 CGGTCAGAGT TGCCCAGGGA AAGCAGCTAT GACATCTACA
    1281 GAGTGCCCAG CAGTCAGAGC ATGGAGGATC GTGGGTACAG
    1321 CCCCGACACG CGTGTGGTCC GCTTCCTCAA GGGCAAGAGC
    1361 ATCGGGCTGC GGCTGGCAGG GGGCAATGAC GTGGGCATCT
    1401 TCGTGTCCGG GGTGCAGGCG GGCAGCCCGG CCGACGGGCA
    1441 GGGCATCCAG GAGGGAGATC AGATTCTGCA GGTGAATGAC
    1481 GTGCCATTCC AGAACCTGAC ACGGGAGGAG GCAGTGCAGT
    1521 TCCTGCTGGG GCTGCCACCA GGCGAGGAGA TGGAGCTGGT
    1561 GACGCAGAGG AAGCAGGACA TTTTCTGGAA AATGGTGCAG
    1601 TCCCGCGTGG GTGACTCCTT CTACATCCGC ACTCACTTTG
    1641 AGCTGGAGCC CAGTCCACCG TCTGGCCTGG GCTTCACCCG
    1681 TGGCGACGTC TTCCACGTGC TGGACACGCT GCACCCCGGC
    1721 CCCGGGCAGA GCCACGCACG AGGAGGCCAC TGGCTGGCGG
    1761 TGCGCATGGG TCGTGACCTG CGGGAGCAAG AGCGGGGCAT
    1801 CATTCCCAAC CAGAGCAGGG CGGAGCAGCT GGCCAGCCTG
    1841 GAAGCTGCCC AGAGGGCCGT GGGAGTCGGG CCCGGCTCCT
    1881 CCGCGGGCTC CAATGCTCGG GCCGAGTTCT GGCGGCTGCG
    1921 GGGTCTTCGT CGAGGAGCCA AGAAGACCAC TCAGCGGAGC
    1961 CGTGAGGACC TCTCAGCTCT GACCCGACAG GGCCGCTACC
    2001 CGCCCTACGA ACGAGTGGTG TTGCGAGAAG CCAGTTTCAA
    2041 GCGCCCGGTA GTGATCCTGG GACCCGTGGC CGACATTGCT
    2081 ATGCAGAAGT TGACTGCTGA GATGCCTGAC CAGTTTGAAA
    2121 TCGCAGAGAC TGTGTCCAGG ACCGACAGCC CCTCCAAGAT
    2161 CATCAAACTA GACACCGTGC GGGTGATTGC AGAAAAAGAC
    2201 AAGCATGCGC TCCTGGATGT GACCCCCTCC GCCATCGAGC
    2241 GCCTCAACTA TGTGCAGTAC TACCCCATTG TGGTCTTCTT
    2281 CATCCCCGAG AGCCGGCCGG CCCTCAAGGC ACTGCGCCAG
    2321 TGGCTGGCGC CTGCCTCCCG CCGCAGCACC CGTCGCCTCT
    2361 ACGCACAAGC CCAGAAGCTG CGAAAACACA GCAGCCACCT
    2401 CTTCACAGCC ACCATCCCTC TGAATGGCAC GAGTGACACC
    2441 TGGTACCAGG AGCTCAAGGC CATCATTCGA GAGCAGCAGA
    2481 CGCGGCCCAT CTGGACGGCG GAAGATCAGC TGGATGGCTC
    2521 CTTGGAGGAC AACCTAGACC TCCCTCACCA CGGCCTGGCC
    2561 GACAGCTCCG CTGACCTCAG CTGCGACAGC CACGTTAACA
    2601 GCGACTACGA GACGGACGGC GAGGGCGGCG CGTACACGGA
    2641 TGGCGAGGGC TACACAGACG GCGAGGGGGG GCCCTACACG
    2681 GATGTGGATG ATGAGCCCCC GGCTCCAGCC CTGGCCCGGT
    2721 CCTCGGAGCC CGTGCAGGCA GATGAGTCCC AGAGCCCGAG
    2761 GGATCGTGGG AGAATCTCGG CTCATCAGGG GGCCCAGGTG
    2801 GACAGCCGCC ACCCCCAGGG ACAGTGGCGA CAGGACAGCA
    2841 TGCGAACCTA TGAACGGGAA GCCCTGAAGA AAAAGTTTAC
    2881 GCGAGTCCGT GATGCGGAGT CCTCCGATGA AGACGGCTAT
    2921 GACTGGGGTC CGGCCACTGA CCTGTGACCT CTCGCAGGCT
    2961 GCCAGCTGGT CCGTCCTCCT TCTCCCTCCC TGGGGCTGGG
    3001 ACTCAGTTTC CCATACAGAA CCCACAACCT TACCTCCCTC
    3041 CGCCTGGTCT TTAATAAACA GAGTATTTTC ACAGC
    • Occludin (OCLN)
  • An amino acid sequence for a human OCLN polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. AAH29886; see also UNIPROT accession no. Q16625) and shown below as SEQ ID NO:7.
  • 1 MSSRPLESPP PYRPDEFKPN HYAPSNDIYG GEMHVRPMLS
    41 QPAYSFYPED EILHFYKWTS PPGVIRILSM LIIVMCIAIF
    81 ACVASTLAWD RGYGTSLLGG SVGYPYGGSG FGSYGSGYGY
    121 GYGYGYGYGG YTDPRAAKGF MLAMAAFCFI AALVIFVTSV
    161 IRSEMSRTRR YYLSVIIVSA ILGIMVFIAT IVYIMGVNPT
    201 AQSSGSLYGS QIYALCNQFY TPAATGLYVD QYSYHYCVVD
    241 PQEAIAIVLG FMIIVAFALI IFFAVKTRRK MDRYDKSNIL
    281 WDKEHIYDEQ PPNVEEWVKN VSAGTQDVPS PPSDYVERVD
    321 SPMAYSSNGK VNDKRFYPES SYKSTPVPEV VQELPLTSPV
    361 DDFRQPRYSS GGNFETPSKR APAKGRAGRS KRTEQDHYET
    401 DYTTGGESCD ELEEDWIREY PPITSDQQRQ LYKRNFDTGL
    441 QEYKSLQSEL DEINKELSRL DKELDDYREE SEEYMAAADE
    481 YNRLKQVKGS ADYKSKKNHC KQLKSKLSHI KKMVGDYDRQ
    521 KT
  • The OCLN gene encodes the OCLN polypeptide with SEQ ID NO:7. The OCLN gene is on chromosome 5 (location NC_000005.10 (69492547 . . . 69558104)). A nucleotide sequence that encodes the OCLN polypeptide with SEQ ID NO:7 is available as NCBI accession no. NG_028291.1. A cDNA sequence encoding the polypeptide having UNIPROT accession no. Q16625 is available as European Nucleotide Archive accession no. U49184, provided below as SEQ ID NO:8.
  • 1 CTCCCGCGTC CACCTCTCCC TCCCTGCTTC CTCTGGCGGA
    41 GGCGGCAGGA ACCGAGAGCC AGGTCCAGAG CGCCGAGGAG
    81 CCGGTCTAGG ACGCAGCAGA TTGGTTTATC TTGGAAGCTA
    121 AAGGGCATTG CTCATCCTGA AGATCAGCTG ACCATTGACA
    161 ATCAGCCATG TCATCCAGGC CTCTTGAAAG TCCACCTCCT
    201 TACAGGCCTG ATGAATTCAA ACCGAATCAT TATGCACCAA
    241 GCAATGACAT ATATGGTGGA GAGATGCATG TTCGACCAAT
    281 GCTCTCTCAG CCAGCCTACT CTTTTTACCC AGAAGATGAA
    321 ATTCTTCACT TCTACAAATG GACCTCTCCT CCAGGAGTGA
    361 TTCGGATCCT GTCTATGCTC ATTATTGTGA TGTGCATTGC
    401 CATCTTTGCC TGTGTGGCCT CCACGCTTGC CTGGGACAGA
    441 GGCTATGGAA CTTCCCTTTT AGGAGGTAGT GTAGGCTACC
    481 CTTATGGAGG AAGTGGCTTT GGTAGCTACG GAAGTGGCTA
    521 TGGCTATGGC TATGGTTATG GCTATGGCTA CGGAGGCTAT
    561 ACAGACCCAA GAGCAGCAAA GGGCTTCATG TTGGCCATGG
    601 CTGCCTTTTG TTTCATTGCC GCGTTGGTGA TCTTTGTTAC
    641 CAGTGTTATA AGATCTGAAA TGTCCAGAAC AAGAAGATAC
    681 TACTTAAGTG TGATAATAGT GAGTGCTATC CTGGGCATCA
    721 TGGTGTTTAT TGCCACAATT GTCTATATAA TGGGAGTGAA
    761 CCCAACTGCT CAGTCTTCTG GATCTCTATA TGGTTCACAA
    801 ATATATGCCC TCTGCAACCA ATTTTATACA CCTGCAGCTA
    841 CTGGACTCTA CGTGGATCAG TATTTGTATC ACTACTGTGT
    881 TGTGGATCCC CAGGAGGCCA TTGCCATTGT ACTGGGGTTC
    921 ATGATTATTG TGGCTTTTGC TTTAATAATT TTCTTTGCTG
    961 TGAAAACTCG AAGAAAGATG GACAGGTATG ACAAGTCCAA
    1001 TATTTTGTGG GACAAGGAAC ACATTTATGA TGAGCAGCCC
    1041 CCCAATGTCG AGGAGTGGGT TAAAAATGTG TCTGCAGGCA
    1081 CACAGGACGT GCCTTCACCC CCATCTGACT ATGTGGAAAG
    1121 AGTTGACAGT CCCATGGCAT ACTCTTCCAA TGGCAAAGTG
    1161 AATGACAAGC GGTTTTATCC AGAGTCTTCC TATAAATCCA
    1201 CGCCGGTTCC TGAAGTGGTT CAGGAGCTTC CATTAACTTC
    1241 GCCTGTGGAT GACTTCAGGC AGCCTCGTTA CAGCAGCGGT
    1281 GGTAACTTTG AGACACCTTC AAAAAGAGCA CCTGCAAAGG
    1321 GAAGAGCAGG AAGGTCAAAG AGAACAGAGC AAGATCACTA
    1361 TGAGACAGAC TACACAACTG GCGGCGAGTC CTGTGATGAG
    1401 CTGGAGGAGG ACTGGATCAG GGAATATCCA CCTATCACTT
    1441 CAGATCAACA AAGACAACTG TACAAGAGGA ATTTTGACAC
    1481 TGGCCTACAG GAATACAAGA GCTTACAATC AGAACTTGAT
    1521 GAGATCAATA AAGAACTCTC CCGTTTGGAT AAAGAATTGG
    1561 ATGACTATAG AGAAGAAAGT GAAGAGTACA TGGCTGCTGC
    1601 TGATGAATAC AATAGACTGA AGCAAGTGAA GGGATCTGCA
    1641 GATTACAAAA GTAAGAAGAA TCATTGCAAG CAGTTAAAGA
    1681 GCAAATTGTC ACACATCAAG AAGATGGTTG GAGACTATGA
    1721 TAGACAGAAA ACATAGAAGG CTGATGCCAA GTTGTTTGAG
    1761 AAATTAAGTA TCTGACATCT CTGCAATCTT CTCAGAAGGC
    1801 AAATGACTTT GGACCATAAC CCCGGAAGCC AAACCTCTGT
    1841 GAGCATCACA AAGTTTTGGT TGCTTTAACA TCATCAGTAT
    1881 TGAAGCATTT TATAAATCGC TTTTGATAAT CAACTGGGCT
    1921 GAACACTCCA ATTAAGGATT TTATGCTTTA AACATTGGTT
    1961 CTTGTATTAA GAATGAAATA CTGTTTGAGG TTTTTAAGCC
    2001 TTAAAGGAAG GTTCTGGTGT GAACTAAACT TTCACACCCC
    2041 AGACGATGTC TTCATACCTA CATGTATTTG TTTGCATAGG
    2081 TGATCTCATT TAATCCTCTC AACCACCTTT CAGATAACTG
    2121 TTATTTATAA TCACTTTTTT CCACATAAGG AAACTGGGTT
    2161 CCTGCAATGA AGTCTCTGAA GTGAAACTGC TTGTTTCCTA
    2201 GCACACACTT TTGGTTAAGT CTGTTTTATG ACTTCATTAA
    2241 TAATAAATTC CCTGGCCTTT CATATTTTAG CTACTATATA
    2281 TGTGATGATC TACCAGCCTC CCTATTTTTT TTCTGTTATA
    2321 TAAATGGTTA AAAGAGGTTT TTCTTAAATA ATAAAGATCA
    2361 TGTAAAAGTA AAAAAAAAA
    • Claudins
  • An amino acid sequence for a human claudin-2 (CLDN2) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no, NP_065117; see also UNIPROT accession no. P57739) and shown below as SEQ ID NO:9.
  • 1 MASLGLQLVG YILGLIGLLG TLVAMLLPSW KTSSYVGASI
    41 VTAVGFSKGL WMECATHSTG ITQCDIYSTL LGLPADIQAA
    81 QAMMVTSSAI SSLACIISVV GMRCTVFCQE SRAKDRVAVA
    121 GGVFFILGGL LGFIPVAWNL HGILRDFYSP LVPDSMKFEI
    161 GEALYLGIIS SLFSLIAGII LCFSCSSQRN RSNYYDAYQA
    201 QPLATRSSPR PGQPPKVKSE FNSYSLTGYV

    The CLDN2 gene encodes the CLDN2 polypeptide with SEQ ID NO:9. The CLDN2 gene is on the X chromosome (location NC_000023.11 (106900164 . . . 106930861). A nucleotide sequence that encodes the CLDN2 polypeptide with SEQ ID NO:9 is available as NCBI accession no. NG_016445.1. A cDNA sequence encoding the polypeptide having NCBI accession no. NM_020384.4 is shown below as SEQ ID NO: 10,
  • 1 GCAGATGGAT TTTGCAAAGC TGTGGTTAAC GATTAGAAAT
    41 CCTTTATCAC CTCAGCCCGT GGCCCCTTGT ACTTCGCTCC
    81 CCTCCCTCAG GATCCCTTTC TCCCTCTCCA GGGGCATCTC
    121 CCCCTCCAAG GCTCTGCAAA GAACTGCCCT GTCTTCTAGA
    161 TGCCTTCTTG AGGCTGCTTG TGGCCACCCA CAGACACTTG
    201 TAAGGAGGAG AGAAGTCAGC CTGGCAGAGA GACTCTGAAA
    241 TGAGGGATTA GAGGTGTTCA AGGAGCAAGA GCTTCAGCCT
    281 GAAGACAAGG GAGCAGTCCC TGAAGACGCT TCTACTGAGA
    321 GGTCTGCCAT GGCCTCTCTT GGCCTCCAAC TTGTGGGCTA
    361 CATCCTAGGC CTTCTGGGGC TTTTGGGCAC ACTGGTTGCC
    401 ATGCTGCTCC CCAGCTGGAA AACAAGTTCT TATGTCGGTG
    441 CCAGCATTGT GACAGCAGTT GGCTTCTCCA AGGGCCTCTG
    481 GATGGAATGT GCCACACACA GCACAGGCAT CACCCAGTGT
    521 GACATCTATA GCACCCTTCT GGGCCTGCCC GCTGACATCC
    561 AGGCTGCCCA GGCCATGATG GTGACATCCA GTGCAATCTC
    601 CTCCCTGGCC TGCATTATCT CTGTGGTGGG CATGAGATGC
    641 ACAGTCTTCT GCCAGGAATC CCGAGCCAAA GACAGAGTGG
    681 CGGTAGCAGG TGGAGTCTTT TTCATCCTTG GAGGCCTCCT
    721 GGGATTCATT CCTGTTGCCT GGAATCTTCA TGGGATCCTA
    761 CGGGACTTCT ACTCACCACT GGTGCCTGAC AGCATGAAAT
    801 TTGAGATTGG AGAGGCTCTT TACTTGGGCA TTATTTCTTC
    841 CCTGTTCTCC CTGATAGCTG GAATCATCCT CTGCTTTTCC
    881 TGCTCATCCC AGAGAAATCG CTCCAACTAC TACGATGCCT
    921 ACCAAGCCCA ACCTCTTGCC ACAAGGAGCT CTCCAAGGCC
    961 TGGTCAACCT CCCAAAGTCA AGAGTGAGTT CAATTCCTAC
    1001 AGCCTGACAG GGTATGTGTG AAGAACCAGG GGCCAGAGCT
    1041 GGGGGGTGGC TGGGTCTGTG AAAAACAGTG GACAGCACCC
    1081 CGAGGGCCAC AGGTGAGGGA CACTACCACT GGATCGTGTC
    1121 AGAAGGTGCT GCTGAGGATA GACTGACTTT GGCCATTGGA
    1161 TTGAGCAAAG GCAGAAATGG GGGCTAGTGT AACAGCATGC
    1201 AGGTTGAATT GCCAAGGATG CTCGCCATGC CAGCCTTTCT
    1241 GTTTTCCTCA CCTTGCTGCT CCCCTGCCCT AAGTCCCCAA
    1281 CCCTCAACTT GAAACCCCAT TCCCTTAAGC CAGGACTCAG
    1321 AGGATCCCTT TGCCCTCTGG TTTACCTGGG ACTCCATCCC
    1361 CAAACCCACT AATCACATCC CACTGACTGA CCCTCTGTGA
    1401 TCAAAGACCC TCTCTCTGGC TGAGGTTGGC TCTTAGCTCA
    1441 TTGCTGGGGA TGGGAAGGAG AAGCAGTGGC TTTTGTGGGC
    1481 ATTGCTCTAA CCTACTTCTC AAGCTTCCCT CCAAAGAAAC
    1521 TGATTGGCCC TGGAACCTCC ATCCCACTCT TGTTATGACT
    1561 CCACAGTGTC CAGACTAATT TGTGCATGAA CTGAAATAAA
    1601 ACCATCCTAC GGTATCCAGG GAACAGAAAG CAGGATGCAG
    1641 GATGGGAGGA CAGGAAGGCA GCCTGGGACA TTTAAAAAAA
    1681 TAAAAATGAA AAAAAAACCC AGAACCCATT TCTCAGGGCA
    1721 CTTTCCAGAA TTCTCTCATA TTTGTGGGCT GGGATCAAGC
    1761 CTGCAGCTTG AGGAAAGCAC AAGGAAAGGA AAGAAGATCT
    1801 GGTGGAAAGC TCAGGTGGCA GCGGACTCTG ACTCCACTGA
    1841 GGAACTGCCT CAGAAGCTGC GATCACAACT TTGGCTGAAG
    1881 CCCCTGCCTC ACTCTAGGGC ACCTGACCTG GCCTCTTGCC
    1921 TAAACCACAA GGCTAAGGGC TATAGACAAT GGTTTCCTTA
    1961 GGAACAGTAA ACCAGTTTTT CTAGGGATGG CCCTTGGCTG
    2001 GGGGATGACA GTGTGGGAGC TGTGGGGTAC TGAGGAAGAC
    2041 ACCATTCCTT GACGGTGTCT AAGAAGCCAG GTGGATGTGT
    2081 GTGGTGGCTC CAGTGGGTGT TTCTACTCTG CCAGTGAGAG
    2121 GCAGCCCCCT AGAAACTCTT CAGGCGTAAT GGAAAATCAG
    2161 CTCAAATGAG ATCAGGCCCC CCCAGGGTCC ACCCACAGAG
    2201 CACTACAGAG CCTCTGAAAG ACCATAGCAC CAAGCGAGCC
    2241 CCTTCAGATT CCCCCACTGT CCATCGGAAG ATGCTCCAGA
    2281 GTGGCTAGAG GGCATCTAAG GGCTCCAGCA TGGCATATCC
    2321 ATGCCCACGG TGCTGTGTCC ATGATCTGAG TGATAGCTGC
    2361 ACTGCTGCCT GGGATTGCAG CTGAGGTGGG AGTGGAGAAT
    2401 GGTTCCCAGG AAGACAGTTC CACCTCTAAG GTCCGAAAAT
    2441 GTTCCCTTTA CCCTGGAGTG GGAGTGAGGG GTCATACACC
    2481 AAAGGTATTT TCCCTCACCA GTCTAGGCAT GACTGGCTTC
    2521 TGAAAAATTC CAGCACACCT CCTCGAACCT CATTGTCAGC
    2561 AGAGAGGGCC CATCTGTTGT CTGTAACATG CCTTTCACAT
    2601 GTCCACCTTC TTGCCATGTT CCAGCTGCTC TCCCAACCTG
    2641 GAAGGCCGTC TCCCCTTAGC CAAGTCCTCC TCAGGCTTGG
    2681 AGAACTTCCT CAGCGTCACC TCCTTCATTG AGCCTTCTCT
    2721 GATCACTCCA TCCCTCTCCT ACCCCTCCCT CCCCCAACCC
    2761 TCAATGTATA AATTGCTTCT TGATGCTTAG CATTCACAAT
    2801 TTTTGATTGA TCGTTATTTG TGTGTGTGTG TCCGATCTCA
    2841 CAAGTATATT GTAAACCCTT CGGTGGGTGG GGGCCATATC
    2881 CTAGACCTCT CTGTATCCCC CAGACTATCT GTAACAGTGC
    2921 CAGGCACACA GTAGGTGATC AATAAACACT TGTTGATTGA
    2961 G
  • An amino acid sequence for a human claudin-5 (CLDN5) isoform 2 polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. NP_001349995; see also UNIPROT accession no. 000501.1) and shown below as SEQ ID NO:11.
  • 1 MGSAALEILG LVLCLVGWGG LILACGLPMW QVTAFLDHNI
    41 VIAQTTWKGL WMSCVVQSTG HMQCKVYDSV LALSTEVQAA
    81 RALTVSAVLL AFVALFVTLA GAQCTTCVAP GPAKARVALT
    121 GGVLYLFCGL LALVPLCWFA NIVVREFYDP SVPVSQKYEL
    161 GAALYIGWAA TALLMVGGCL LCCGAWVCTG RPDLSFPVKY
    201 SAPRRPTATG DYDKKNYV

    The CLDN5 gene encodes the CLDN5 polypeptide with SEQ ID NO:11. The CLDN5 gene is on chromosome 22 (location NC_000022.11 (19523024 . . . 19525337, complement)). A cDNA sequence that encodes the CLDN5 polypeptide with SEQ ID NO: 11 is available as NCBI accession no. NM_001363066, shown below as SEQ ID NO: 12.
  • 1 GGCAGACCCA GGAGGTGCGA CAGACCCGCG GGGCAAACGG
    41 ACTGGGGCCA AGAGCCGGGA GCGCGGGCGC AAAGGCACCA
    81 GGGCCCGCCC AGGGCGCCGC GCAGCACGGC CTTGGGGGTT
    121 CTGCGGGCCT TCGGGTGCGC GTCTCGCCTC TAGCCATGGG
    161 GTCCGCAGCG TTGGAGATCC TGGGCCTGGT GCTGTGCCTG
    201 GTGGGCTGGG GGGGTCTGAT CCTGGCGTGC GGGCTGCCCA
    241 TGTGGCAGGT GACCGCCTTC CTGGACCACA ACATCGTGAC
    281 GGCGCAGACC ACCTGGAAGG GGCTGTGGAT GTCGTGCGTG
    321 GTGCAGAGCA CCGGGCACAT GCAGTGCAAA GTGTACGACT
    361 CGGTGCTGGC TCTGAGCACC GAGGTGCAGG CGGCGCGGGC
    401 GCTCACCGTG AGCGCCGTGC TGCTGGCGTT CGTTGCGCTC
    441 TTCGTGACCC TGGGGGGCGC GCAGTGCACC ACCTGCGTGG
    481 CCCCGGGCCC GGCCAAGGCG CGTGTGGCCC TCACGGGAGG
    521 CGTGCTCTAC CTGTTTTGCG GGCTGCTGGC GCTCGTGCCA
    561 CTCTGCTGGT TCGCCAACAT TGTCGTCCGC GAGTTTTACG
    601 ACCCGTCTGT GCCCGTGTCG CAGAAGTACG AGCTGGGCGC
    641 AGCGCTGTAC ATCGGCTGGG CGGCCACCGC GCTGCTCATG
    681 GTAGGCGGCT GCCTCTTGTG CTGCGGCGCC TGGGTCTGCA
    721 CCGGCCGTCC CGACCTCAGC TTCCCCGTGA AGTACTCAGC
    761 GCCGCGGCGG CCCACGGCCA CCGGCGACTA CGACAAGAAG
    801 AACTACGTCT GAGGGCGCTG GGCACGGCCG GGCCCCTCCT
    841 GCCAGCCACG CCTGCGAGGC GTTGGATAAG CCTGGGGAGC
    881 CCCGCATGGA CCGCGGCTTC CGCCGGGTAG CGCGGCGCGC
    921 AGGCTCCTCG GAACGTCCGG CTCTGCGCCC CGACGCGGCT
    961 CCTGGATCCG CTCCTGCCTG CGCCCGCAGC TGACCTTCTC
    1001 CTGCCACTAG CCCGGCCCTG CCCTTAACAG ACGGAATGAA
    1041 GTTTCCTTTT CTGTGCGCGG CGCTGTTTCC ATAGGCAGAG
    1081 CGGGTGTCAG ACTGAGGATT TCGCTTCCCC TCCAAGACGC
    1121 TGGGGGTCTT GGCTGCTGCC TTACTTCCCA GAGGCTCCTG
    1161 CTGACTTCGG AGGGGCGGAT GCAGAGCCCA GGGCCCCCAC
    1201 CGGAAGATGT GTACAGCTGG TCTTTACTCC ATCGGCAGGG
    1241 CCCGAGCCCA GGGACCAGTG ACTTGGCCTG GACCTCCCGG
    1281 TCTCACTCCA GCATCTCCCC AGGCAAGGCT TGTGGGCACC
    1321 GGAGCTTGAG AGAGGGCGGG AGTGGGAAGG CTAAGAATCT
    1361 GCTTAGTAAA TGGTTTGAAC TCTC
  • An amino acid sequence for a human claudin-6 (CLDN6) polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. NP_067018; see also UNIPROT accession no. P56747.2) and shown below as SEQ ID NO:13.
  • 1 MASAGMQILG VVLTLLGWVN GLVSCALPMW KVTAFIGNSI
    41 VVAQVVWEGL WMSCVVQSTG QMQCKVYDSL LALPQDLQAA
    81 RALCVIALLV ALFGLIVYLA GAKCTTCVEE KDSKARLVLT
    121 SGIVFVISGV LTLIPVCWTA HAIIRDFYNP LVAEAQKREL
    161 GASLYLGWAA SGLLLLGGGL LCCTCPSGGS QGPSHYMARY
    181 STSAPAISRG PSEYPTKNYV

    The CLDN6 gene encodes the CLDN6 polypeptide with SEQ ID NO:13. The CLDN6 gene is on chromosome 16 (location NC_000016.10 (3014712 . . . 3018183, complement)). A cDNA sequence that encodes the CLDN6 polypeptide with SEQ ID NO: 13 is available as NCBI accession no. NM_021195.5, shown below as SEQ ID NO: 14.
  • 1 ACTCGGCCTA GGAATTTCCC TTATCTCCTT CGCAGTGCAG
    41 CTCCTTCAAC CTCGCCATGG CCTCTGCCGG AATGCAGATC
    81 CTGGGAGTCG TCCTGACACT GCTGGGCTGG GTGAATGGCC
    121 TGGTCTCCTG TGCCCTGCCC ATGTGGAAGG TGACCGCTTT
    161 CATCGGCAAC AGCATCGTGG TGGCCCAGGT GGTGTGGGAG
    201 GGCCTGTGGA TGTCCTGCGT GGTGCAGAGC ACCGGCCAGA
    241 TGCAGTGCAA GGTGTACGAC TCACTGCTGG CGCTGCCACA
    281 GGACCTGCAG GCTGCACGTG CCCTCTGTGT CATCGCCCTC
    321 CTTGTGGCCC TGTTCGGCTT GCTGGTCTAC CTTGCTGGGG
    361 CCAAGTGTAC CACCTGTGTG GAGGAGAAGG ATTCCAAGGC
    401 CCGCCTGGTG CTCACCTCTG GGATTGTCTT TGTCATCTCA
    441 GGGGTCCTGA CGCTAATCCC CGTGTGCTGG ACGGCGCATG
    481 CCATCATCCG GGACTTCTAT AACCCCCTGG TGGCTGAGGC
    521 CCAAAAGCGG GAGCTGGGGG CCTCCCTCTA CTTGGGCTGG
    561 GCGGCCTCAG GCCTTTTGTT GCTGGGTGGG GGGTTGCTGT
    601 GCTGCACTTG CCCCTCGGGG GGGTCCCAGG GCCCCAGCCA
    641 TTACATGGCC CGCTACTCAA CATCTGCCCC TGCCATCTCT
    681 CGGGGGCCCT CTGAGTACCC TACCAAGAAT TACGTCTGAC
    721 GTGGAGGGGA ATGGGGGCTC CGCTGGCGCT AGAGCCATCC
    761 AGAAGTGGCA GTGCCCAACA GCTTTGGGAT GGGTTCGTAC
    801 CTTTTGTTTC TGCCTCCTGC TATTTTTCTT TTGACTGAGG
    841 ATATTTAAAA TTCATTTGAA AACTGAGCCA AGGTGTTGAC
    881 TCAGACTCTC ACTTAGGCTC TGCTGTTTCT CACCCTTGGA
    921 TGATGGAGCC AAAGAGGGGA TGCTTTGAGA TTCTGGATCT
    961 TGACATGCCC ATCTTAGAAG CCAGTCAAGC TATGGAACTA
    1001 ATGCGGAGGC TGCTTGCTGT GCTGGCTTTG CAACAAGACA
    1041 GACTGTCCCC AAGAGTTCCT GCTGCTGCTG GGGGCTGGGC
    1081 TTCCCTAGAT GTCACTGGAC AGCTGCCCCC CATCCTACTC
    1121 AGGTCTCTGG AGCTCCTCTC TTCACCCCTG GAAAAACAAA
    1161 TGATCTGTTA ACAAAGGACT GCCCACCTCC GGAACTTCTG
    1201 ACCTCTGTTT CCTCCGTCCT GATAAGACGT CCACCCCCCA
    1241 GGGCCAGGTC CCAGCTATGT AGACCCCCGC CCCCACCTCC
    1281 AACACTGCAC CCTTCTGCCC TGCCCCCCTC GTCTCACCCC
    1321 CTTTACACTC ACATTTTTAT CAAATAAAGC ATGTTTTGTT
    1361 AGTGCA
  • An amino acid sequence for a human claudin-7 (CLDN7) isoform 1 polypeptide is available in the NCBI and UNIPROT databases (NCBI accession no. NP_001298; see also UNIPROT accession no. 095471.4) and shown below as SEQ ID NO: 15.
  • 1 MANSGLQLLG FSMALLGWVG LVACTAIPQW QMSSYAGDNI
    41 ITAQAMYKGL WMDCVTQSTG MMSCKMYDSV LALSAALQAT
    81 RALMVVSLVL GFLAMFVATM GMKCTRCGGD DKVKKARIAM
    121 GGGIIFIVAG LAALVACSWY GHQIVTDFYN PLIPTNIKYE
    161 FGPAIFIGWA GSALVILGGA LLSCSCPGNE SKAGYRVPRS
    201 YPKSNSSKEY V

    The CLDN7 gene encodes the CLDN7 polypeptide with SEQ ID NO:15. The CLDN7 gene is on chromosome 17 (NC_000017.11 (7259903 . . . 7263213, complement). A cDNA sequence that encodes the CLDN7 polypeptide with SEQ ID NO: 15 is available as NCBI accession no. NM_001307.6, shown below as SEQ ID NO:16.
  • 1 GCCCGCACCT GCTGGCTCAC CTCCGAGCCA CCTCTGCTGC
    41 GCACCGCAGC CTCGGACCTA CAGCCCAGGA TACTTTGGGA
    81 CTTGCCGGCG CTCAGAAACG CGCCCAGACG GCCCCTCCAC
    121 CTTTTGTTTG CCTAGGGTCG CCGAGAGCGC CCGGAGGGAA
    161 CCGCCTGGCC TTCGGGGACC ACCAATTTTG TCTGGAACCA
    201 CCCTCCCGGC GTATCCTACT CCCTGTGCCG CGAGGCCATC
    241 GCTTCACTGG AGGGGTCGAT TTGTGTGTAG TTTGGTGACA
    281 AGATTTGCAT TCACCTGGCC CAAACCCTTT TTGTCTCTTT
    321 GGGTGACCGG AAAACTCCAC CTCAAGTTTT CTTTTGTGGG
    361 GCTGCCCCCC AAGTGTCGTT TGTTTTACTG TAGGGTCTCC
    401 CCGCCCGGCG CCCCCAGTGT TTTCTGAGGG CGGAAATGGC
    441 CAATTCGGGC CTGCAGTTGC TGGGCTTCTC CATGGCCCTG
    481 CTGGGCTGGG TGGGTCTGGT GGCCTGCACC GCCATCCCGC
    521 AGTGGCAGAT GAGCTCCTAT GCGGGTGACA ACATCATCAC
    561 GGCCCAGGCC ATGTACAAGG GGCTGTGGAT GGACTGCGTC
    601 ACGCAGAGCA CGGGGATGAT GAGCTGCAAA ATGTACGACT
    641 CGGTGCTCGC CCTGTCCGCG GCCTTGCAGG CCACTCGAGC
    681 CCTAATGGTG GTCTCCCTGG TGCTGGGCTT CCTGGCCATG
    721 TTTGTGGCCA CGATGGGCAT GAAGTGCACG CGCTGTGGGG
    761 GAGACGACAA AGTGAAGAAG GCCCGTATAG CCATGGGTGG
    801 AGGCATAATT TTCATCGTGG CAGGTCTTGC CGCCTTGGTA
    841 GCTTGCTCCT GGTATGGCCA TCAGATTGTC ACAGACTTTT
    881 ATAACCCTTT GATCCCTACC AACATTAAGT ATGAGTTTGG
    921 CCCTGCCATC TTTATTGGCT GGGCAGGGTC TGCCCTAGTC
    961 ATCCTGGGAG GTGCACTGCT CTCCTGTTCC TGTCCTGGGA
    1001 ATGAGAGCAA GGCTGGGTAC CGTGTACCCC GCTCTTACCC
    1041 TAAGTCCAAC TCTTCCAAGG AGTATGTGTG ACCTGGGATC
    1081 TCCTTGCCCC AGCCTGACAG GCTATGGGAG TGTCTAGATG
    1121 CCTGAAAGGG CCTGGGGCTG AGCTCAGCCT GTGGGCAGGG
    1161 TGCCGGACAA AGGCCTCCTG GTCACTCTGT CCCTGCACTC
    1201 CATGTATAGT CCTCTTGGGT TGGGGGTGGG GGGGTGCCGT
    1241 TGGTGGGAGA GACAAAAAGA GGGAGAGTGT GCTTTTTGTA
    1281 CAGTAATAAA AAATAAGTAT TGGGAAGCAG GCTTTTTTCC
    1321 CTTCAGGGCC TCTGCTTTCC TCCCGTCCAG ATCCTTGCAG
    1361 GGAGCTTGGA ACCTTAGTGC ACCTACTTCA GTTCAGAACA
    1401 CTTAGCACCC CACTGACTCC ACTGACAATT GACTAAAAGA
    1441 TGCAGGTGCT CGTATCTCGA CATTCATTCC CACCCCCCTC
    1481 TTATTTAAAT AGCTACCAAA GTACTTCTTT TTTAATAAAA
    1521 AAATAAAGAT TTTTATTAGG TA
    • Variants and Modified Tight Junction Proteins
  • Zonula occludens, OCLN, and claudin (CLDN) sequences can vary amongst the human population. Variants can include codon variations and/or conservative amino acid changes. Zonula occludens (TJP), OCLN, and claudin (CLDN) nucleotide and protein sequences can also include non-conservative variations. For example, the zonula occludens (TJP), OCLN, and claudin (CLDN) nucleic acids or proteins can have at least 85% sequence identity and/or complementary, or at least 90% sequence identity and/or complementary, or at least 95% sequence identity and/or complementarity, or at least 96% sequence identity and/or complementarity, or at least 97% sequence identity and/or complementarity, or at least 98% sequence identity and/or complementarity, or at least 99% sequence identity and/or complementarity to any of the Zonula occludens (TJP), OCLN, and claudin (CLDN) nucleic acid or protein sequences described herein.
  • As illustrated herein, inhibition or loss of function of tight junction gene products (e.g., ZO1) can facilitate conversion of hiPSCs to primordial germ cells. Loss of function modifications to tight junction genes and gene products can be introduced by any method. Other possible methods of silencing/disrupting tight junction genes include using short interfering RNA (siRNA), using CRISPR to knockout or mutate a tight junction gene, or simply using chemical inhibition (EDTA or other calcium chelators, for example).
  • For example, genetic loci encoding tight junction proteins can be modified in human iPSC lines by deletion, insertion, or substitution. A variety of methods and inhibitors can be used to reduce the function of these tight junction proteins. For example, the hiPSCs or iMeLCs can be contacted with CRISPRi ribonucleoprotein (RNP) complexes, inhibitory nucleic acids, expression vectors, virus-like particles (VLP), CRISPR-related, and combinations thereof that target the tight junction genes or mRNAs.
  • The CRISPR-Cas9 genome-editing system can be used to delete modify tight junction coding regions or regulatory elements. A single guide RNA (sgRNA) can be used to recognize one or more target sequence in a subject's genome, and a nuclease can act as a pair of scissors to cleave a single-strand or a double-strand of genomic DNA. Mutations in the genome that are near the cleavage site can be introduced by an endogenous Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR) pathway. Hence, the guide RNAs guide the nuclease to cleave the targeted tight junction genomic site for deletion and/or modification by endogenous mechanisms.
  • The Cas system can recognize any sequence in the genome that matches 20 bases of a gRNA. However, each gRNA should also be adjacent to a “Protospacer Adjacent Motif” (PAM), which is invariant for each type of Cas protein, because the PAM binds directly to the Cas protein. See Doudna et al., Science 346 (6213): 1077, 1258096 (2014); and Jinek et al., Science 337:816-21 (2012). Hence, the guide RNAs can have a PAM site sequence that can be bound by a Cas protein.
  • When the Cas system was first described for Cas9, with a “NGG” PAM site, the PAM was somewhat limiting in that it required a GG in the right orientation to the site to be targeted. Different Cas9 species have now been described with different PAM sites. See Jinek et al., Science 337:816-21 (2012); Ran et al., Nature 520:186-91 (2015); and Zetsche et al., Cell 163:759-71 (2015). In addition, mutations in the PAM recognition domain (Table 1) have increased the diversity of PAM sites for SpCas9 and SaCas9. See Kleinstiver et al., Nat Biotechnol 33:1293-1298 (2015); and Kleinstiver et al., Nature 523:481-5 (2015). The following are examples of PAM sites.
  • TABLE 1
    PAM Sequences
    Cas Nuclease PAM Sequence
    SpCas9 NGG
    SpCas9 VRER variant NGCG
    SpCas9 EQR variant NGAG
    SpCas9 VQR variant NGAN or NGNG
    SaCas9 NNGRRT
    SaCas9, KKH variant NNNRRT
    FnCas2 (Cpf1) TTN
    DNA annotations:
    N = A, C, T or G;
    R = Purine, A or G
    Note that the guide RNAs for SpCas9 and SaCas9 cover 20 bases in the 5′direction of the PAM site, while for FnCas2 (Cpf1) the guide RNA covers 20 bases to 3′ of the PAM.
  • There are a number of different types of nucleases and systems that can be used for gene editing. The nuclease employed can in some cases be any DNA binding protein with nuclease activity. Examples of nuclease include Streptococcus pyogenes Cas (SpCas9) nucleases, Staphylococcus aureus Cas9 (SpCas9) nucleases, Francisella novicida Cas2 (FnCas2, also called dFnCpf1) nucleases, Zinc Finger Nucleases (ZFN), Meganuclease, Transcription activator-like effector nucleases (TALEN), Fok-I nucleases, any DNA binding protein with nuclease activity, any DNA binding protein bound to a nuclease, or any combinations thereof. However, the CRISPR-Cas systems are generally the most widely used. In some cases, the nuclease is therefore a Cas nuclease.
  • CRISPR-Cas systems are generally divided into two classes. The class 1 system contains types I, III and IV, and the class 2 system contains types II, V, and VI. The class 1 CRISPR-Cas system uses a complex of several Cas proteins, whereas the class 2 system only uses a single Cas protein with multiple domains. The class 2 CRISPR-Cas system is usually preferable for gene-engineering applications because of its simplicity and ease of use.
  • A variety of Cas nucleases can be employed in the methods described herein. Three species that have been best characterized are provided as examples. The most commonly used Cas nuclease is a Streptococcus pyogenes Cas9, (SpCas9). More recently described forms of Cas include Staphylococcus aureus Cas9 (SaCas9) and Francisella novicida Cas2 (FnCas2, also called FnCpf1). Jinek et al., Science 337:816-21 (2012); Qi et al., Cell 152:1173-83 (2013); Ran et al., Nature 520:186-91 (2015); Zetsche et al., Cell 163:759-71 (2015).
  • Inhibitory nucleic acids can be used to reduce the expression and/or translation of tight junction. Such inhibitory nucleic acids can specifically bind to tight junction nucleic acids, including nascent RNAs, that encode a tight junction protein. Anti-sense oligonucleotides have been used to silence regulatory elements as well.
  • An inhibitory nucleic acid can have at least one segment that will hybridize to tight junction nucleic acid under intracellular or stringent conditions. The inhibitory nucleic acid can reduce processing, expression, and/or translation of a nucleic acid encoding tight junction. An inhibitory nucleic acid may hybridize to a genomic DNA, a messenger RNA, nascent RNA, or a combination thereof. An inhibitory nucleic acid may be incorporated into a plasmid vector or viral DNA. It may be single stranded or double stranded, circular, or linear.
  • An inhibitory nucleic acid can be a polymer of ribose nucleotides (RNAi) or deoxyribose nucleotides having more than 13 nucleotides in length. An inhibitory nucleic acid may include naturally-occurring nucleotides; synthetic, modified, or pseudo-nucleotides such as phosphorothiolates; as well as nucleotides having a detectable label such as P32, biotin or digoxigenin. An inhibitory nucleic acid can reduce the expression, processing, and/or translation of a tight junction nucleic acid.
  • Such an inhibitory nucleic acid may be completely complementary to a segment of tight junction nucleic acid (e.g., a tight junction mRNA or tight junction nascent transcript).
  • An inhibitory nucleic acid can hybridize to a tight junction nucleic acid under intracellular conditions or under stringent hybridization conditions and is sufficient to inhibit expression of a tight junction nucleic acid. Intracellular conditions refer to conditions such as temperature, pH and salt concentrations typically found inside a cell, e.g. a target cell described herein.
  • Generally, stringent hybridization conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C. lower than the thermal melting point of the selected sequence, depending upon the desired degree of stringency as otherwise qualified herein. Inhibitory oligonucleotides that comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides that are precisely complementary to a tight junction coding or flanking sequence, can each be separated by a stretch of contiguous nucleotides that are not complementary to adjacent coding sequences, and such an inhibitory nucleic acid can still inhibit the function of a tight junction nucleic acid. In general, each stretch of contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences may be 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an inhibitory nucleic acid hybridized to a sense nucleic acid to estimate the degree of mismatching that will be tolerated for inhibiting expression of a particular target nucleic acid. Inhibitory nucleic acids of the invention include, for example, a short hairpin RNA, a small interfering RNA, a ribozyme, or an antisense nucleic acid molecule.
  • The inhibitory nucleic acid molecule may be single (e.g., an antisense oligonucleotide) or double stranded (e.g., a siRNA) and may function in an enzyme-dependent manner or by steric blocking. Inhibitory nucleic acid molecules that function in an enzyme-dependent manner include forms dependent on RNase H activity to degrade target mRNA. These include single-stranded DNA, RNA, and phosphorothioate molecules, as well as the double-stranded RNAi/siRNA system that involves target mRNA recognition through sense-antisense strand pairing followed by degradation of the target mRNA by the RNA-induced silencing complex. Steric blocking inhibitory nucleic acids, which are RNase-H independent, interfere with gene expression or other mRNA-dependent cellular processes by binding to a target mRNA and getting in the way of other processes. Steric blocking inhibitory nucleic acids include 2′-O alkyl (usually in chimeras with RNase-H dependent antisense), peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholino antisense.
  • Small interfering RNAs (siRNAs), for example, may be used to specifically reduce tight junction processing or translation such that production of the encoded polypeptide is reduced. SiRNAs mediate post-transcriptional gene silencing in a sequence-specific manner. See, for example, website at invitrogen.com/site/us/en/home/Products-and-Services/Applications/mai.html. Once incorporated into an RNA-induced silencing complex, siRNA can mediate cleavage of the homologous endogenous mRNA transcript by guiding the complex to the homologous mRNA transcript, which is then cleaved by the complex. The siRNA may be homologous to any region of the tight junction mRNA transcript. The region of homology may be 50 nucleotides or less, 30 nucleotides or less in length, such as less than 25 nucleotides, or for example about 21 to 23 nucleotides in length. SiRNA is typically double stranded and may have two-nucleotide 3′ overhangs, for example, 3′ overhanging UU dinucleotides. Methods for designing siRNAs are available, see, for example, Elbashir et al. Nature 411:494-498 (2001); Harborth et al. Antisense Nucleic Acid Drug Dev. 13:83-106 (2003).
  • The pSuppressorNeo vector for expressing hairpin siRNA, commercially available from IMGENEX (San Diego, California), can be used to make siRNA or shRNA for inhibiting tight junction expression. The construction of the siRNA or shRNA expression plasmid involves the selection of the target region of the mRNA, which can be a trial-and-error process. However, Elbashir et al. have provided guidelines that appear to work ˜80% of the time. Elbashir, S. M., et al., Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 2002. 26 (2): p. 199-213. Accordingly, for synthesis of synthetic siRNA or shRNA, a target region may be selected preferably 50 to 100 nucleotides downstream of the start codon. The 5′ and 3′ untranslated regions and regions close to the start codon should be avoided as these may be richer in regulatory protein binding sites. As siRNA can begin with AA, have 3′ UU overhangs for both the sense and antisense siRNA strands, and have an approximate 50% G/C content. An example of a sequence for a synthetic siRNA or shRNA is 5′-AA (N19) UU, where N is any nucleotide in the mRNA sequence and should be approximately 50% G-C content. The selected sequence(s) can be compared to others in the human genome database to minimize homology to other known coding sequences (e.g., by Blast search, for example, through the NCBI website).
  • Inhibitory nucleic acids (e.g., siRNAs, and/or anti-sense oligonucleotides) may be chemically synthesized, created by in vitro transcription, or expressed from an expression vector or a PCR expression cassette. See, e.g., website at invitrogen.com/site/us/en/home/Products-and-Services/Applications/rai.html.
  • When an siRNA is expressed from an expression vector or a PCR expression cassette, the insert encoding the siRNA may be expressed as an RNA transcript that folds into an siRNA hairpin or a shRNA. Thus, the RNA transcript may include a sense siRNA sequence that is linked to its reverse complementary antisense siRNA sequence by a spacer sequence that forms the loop of the hairpin as well as a string of U's at the 3′ end. The loop of the hairpin may be of any appropriate lengths, for example, 3 to 30 nucleotides in length, or about 3 to 23 nucleotides in length, and may include various nucleotide sequences including for example, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC. SiRNAs also may be produced in vivo by cleavage of double-stranded RNA introduced directly or via a transgene or virus. Amplification by an RNA-dependent RNA polymerase may occur in some organisms.
  • An inhibitory nucleic acid such as a short hairpin RNA siRNA or an antisense oligonucleotide may be prepared using methods such as by expression from an expression vector or expression cassette that includes the sequence of the inhibitory nucleic acid. Alternatively, it may be prepared by chemical synthesis using naturally-occurring nucleotides, modified nucleotides, or any combinations thereof. In some embodiments, the inhibitory nucleic acids are made from modified nucleotides or non-phosphodiester bonds, for example, that are designed to increase biological stability of the inhibitory nucleic acid or to increase intracellular stability of the duplex formed between the inhibitory nucleic acid and the target tight junction nucleic acid.
    • Differentiation of Primordial Germ Cells
  • Primordial germ cells can be differentiated into mature germ cells, including functional oocyte and sperm by in vitro culture or by implantation in a selected subject. A variety of differentiation methods can be used including those described in U.S. patent application No. 20180251729. Previous studies in mice illustrate methods for generating functional male and female gametes from PGCLCs in vivo, which can then be used to produce live offspring through IVF (Hayashi et al., Cell 2011) (Hayashi et al., Science 2013) (Zhou et al., Science 2013). Xenogenic and allogenic transplantation of primordial germ cells into the ovarian bursa, seminiferous tubules of the testes, or under the kidney capsule of mice successfully induced meiosis in the transplanted PGCs, establishing a proof-of-concept method for PGC maturation that potentially circumvents the need for developing an in vitro protocol to mature human PGCs (Hayama et al., Biol. Reprod 2014) (Matoba et al., Biol. Reprod 2011) (Qing et al., Hum. Reprod. 2008). Additionally, it has recently been shown that human female PGCs can be matured to oogonia by xenogeneic culture with mouse embryonic ovarian somatic cells (Yamashiro et al., Science 2020).
  • The following Examples illustrate some of the experiments that were performed in the development of the invention.
    • Example 1: Methods
  • This Example describes some of the materials and methods used in developing the invention.
    • Cell Culture
  • Human iPSC lines were derived from the male Allen Institute WTC-LMNB1-meGFP line (Cell Line ID: AICS-0013 cl.210, passage 32) obtained from Coriel, and/or the female WTB CRISPRi-Gen1B line (Gladstone Stem Cell Core, passage 40) provided by Dr. Bruce Conklin's lab. For routine culture, human induced pluripotent stem cells (hiPSCs) were grown feeder-free on growth factor reduced Matrigel (BD Biosciences) and fed daily with mTESR1 medium (Stem Cell Technologies). Cells were passaged every 3-4 days with Accutase (Stem Cell Technologies) and seeded at a density of 12,000 cells/cm2. ROCK inhibitor Y-276932 (10 uM; Selleckchem) was added to the media to promote cell survival after passaging. All generated cell lines were karyotyped prior to expansion and confirmed as normal cells both by Cell Line Genetics and by using the hPSC Genetic Analysis Kit (Stem Cell Technologies Cat. #07550). The cells were also regularly tested for mycoplasma using a MycoAlert Mycoplasma Detection Kit (Lonza).
    • Generation of CRISPi Lines
  • Knockdown (KD) of ZO1 in hiPSC lines was achieved using a doxycycline (DOX) inducible CRISPR interference (CRISPRi) system, which included two components. First, a dCas9-KRAB repressor driven by a Tet-on-3G promoter was knocked in into the AAVS1 safe harbor locus and expressed only under DOX treatment described by Mandegar et al. Cell Stem Cell 18, 541-553 (2016) (FIG. 1A). Second, a constitutively expressed guide RNA (gRNA) was used that targets the transcriptional start site of a gene (FIG. 1A). Briefly, about 2 million WTC or WTB derived cells were nucleofected with the knockin vector (5 ug) along with TALENS targeting the AAVS1 locus (2 ug) and cultured in mTESR1 and ROCK inhibitor Y-276932 (10 uM). Knockin selection was performed with Genticin (100 ug/mL Life Technologies) over the course of 10 days, and a clonal population was generated through colony picking under the EVOS picking microscope (Life Technologies).
  • To generate the ZO1-WTC line, four CRISPRi gRNAs were designed to bind within 150 bp of the transcription start site of ZO1 and cloned into the gRNA-CKB vector at the BsmB1 restriction site, following the protocol described in Mandegar et al. (2016). The sequences of the ZO1 guide RNAs that were used are shown in Table 2 below.
  • TABLE 2
    CRISPRi gRNAs
    Guide RNA Location
    (gRNA) Target to TSS Sequence
    ZO1_1  67 CCGGTTCCCGGGAAGTTACG
    (SEQ ID NO: 17)
    ZO1_2 271 CAGGGGGAGGGAATTCAACT
    (SEQ ID NO: 18)
    ZO1_3 147 CTTTCGCAGCCCGGCCACGT
    (SEQ ID NO: 19)
    ZO1_4  76 GGGAAGTTACGTGGCGAAGC
    (SEQ ID NO: 20)
  • Vectors containing each gRNA sequence were individually nucleofected into the WTC-LMNB1-mEGFP line (containing the CRISPRi-KRAB construct) using the Human Stem Cell Nucleofector Kit 1 solution with the Amaxa nucleofector 2b device (Lonza). Nucleofected cells were subsequently seeded at a density of 8,000 cells/cm2 and recovered in mTESR1 media supplemented with ROCK inhibitor Y-276932 (10 uM) for two days. Guide selection was performed with blasticidin (10 ug/mL, ThermoFisher Scientific) for seven days, and clonal populations were generated through colony picking. Knockdown efficiency was evaluated through exposure to doxycycline (2 uM) for five days, after which mRNA was isolated, and relative levels of ZO1 were assessed through qPCR. Levels of ZO1 were normalized to copy numbers from the same line without CRISPRi induction.
  • The most effective was guide selected (ZO1_1 gRNA; CCGGTTCCCGGGAAGTTACG (SEQ ID NO:17)). After validation, this guide was subsequently introduced into the WTB CRISPRi-Gen1B line, which was selected and validated using the same methods.
    • PGCLC Induction Using BMP-4 Colony Differentiation
  • To determine changes in proportions of germ lineage fates in Control (ZWT, ˜ DOX) and ZO1 KD (ZKD, +DOX) hiPSCs, unconfined colonies from each condition were treated with BMP-4 (50 ng/mL) in mTESR1 culture medium for 48 hours. The ZO1 knockdown cells were then stained for appropriate germ lineage markers. Note that for these experiments involving evaluation of the ability of monolayers and cell colonies to form PGCLCs, only ZO1 knockdown cells were used (because wild type cells in monolayers and colonies do not form PGCLCs without basolateral exposure to BMP).
  • Uniform colonies (˜100 ZO1 KD cells/colony) were achieved by seeding about 10,000 cells in mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM) from each condition into 400 by 400 mm PDMS microwell inserts (containing approximately 975 microwells) and force aggregating the cells through centrifugation at 200RCF for 3 minutes, using protocols adapted from those by Hookway et al., 2016; Ungrin et al., 2008 (FIG. 2B). After 18 hours, the aggregates were transferred in mTESR1 to Matrigel-coated 96 well plates at a density of approximately 10 aggregates/well. The cells were then allowed to attach and flatten into two dimensional (2D) colonies over the course of 24 hours prior to stimulation with BMP-4.
  • PGCLC Induction with BMP-4 Monolayer Differentiation
  • ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells were seeded in mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM) into 96 well plates at a density between 100-350 cells/mm2. The following day, the cells were fed with 100 ul-200 ul of mTESR1. On day 2, the cells were induced with BMP-4 (50 ng/ml) in mTESR1. At 48 and 72 hours after induction with BMP-4, the cells were fixed prior to staining for PGCLC and other somatic lineage markers. mRNA was collected from the 48 hour timepoint for qPCR analysis, the primers used for qPCR are listed in Tables 3-4.
  • TABLE 3
    Primers for Pluripotency Genetic Markers
    Gene First Primer Second Primer
    OCT4 ATGCATTCAAACTG AACTTCACCTTCCCTC
    AGGTGCCT (SEQ ID NO: 21) CAACCA (SEQ ID NO: 22)
    NANOG CCCAAAGGCAAACAA AGCTGGGTGGAAGAGA
    CCCACTTCT (SEQ ID NO: 23) ACACAGTT (SEQ ID NO: 24)
    DPPA3 TGTTACTCGGCGGAG GATCCCATCCATTAGA
    TTCGTAC (SEQ ID NO: 25) CACGCAG (SEQ ID NO: 26)
    SOX2 AACCAGCGCATGGAC CGAGCTGGTCATGGA
    AGTTA (SEQ ID NO: 27) GTTGT (SEQ ID NO: 28)
    PRDM14 CCTTGTGTGGTATGG CTTTCACATCTGTAGC
    AGACTGC (SEQ ID NO: 29) CTTCTGC (SEQ ID NO: 30)
    OTX2 GGAAGCACTGTTTGCC CTGTTGTTGGCGGCA
    AAGACC (SEQ ID NO: 31) CTTAGCT (SEQ ID NO: 32)
    SOX11 GCTGAAGGACAGCGA GGGTCCATTTTGGGC
    GAAGATC (SEQ ID NO: 33) TTTTTCCG (SEQ ID NO: 34)
    18S CTCTAGTGATCCCTG ACTCGCTCCACCTCA
    AGAAGTTCC (SEQ ID NO: 35) TCCTC (SEQ ID NO: 36)
  • TABLE 4
    Somatic/Germ Lineage Genetic Linkages
    Gene First Primer Second Primer
    ZO1 GCAGCTAGCCAGTGTA GCCTCAGAAATCCAGC
    CAGTATAC (SEQ ID NO: 37) TTCTCGAA (SEQ ID NO: 38)
    T TTTCCAGATGGTGAGA CCGATGCCTCAACTCT
    GCCG (SEQ ID NO: 39) CCAG (SEQ ID NO: 40)
    NANOS3 CCCGAAACTCGGCAG AAGGCTCAGACTTCCC
    GCAAGA (SEQ ID NO: 41) GGCAC (SEQ ID NO: 42)
    BLIMP1 CGGGGAGAATGTGGACT CTGGAGTTACACTTGG
    GGGTAGAG (SEQ ID NO: 43) GGGCAGC (SEQ ID NO: 44)
    SOX17 GAGCCAAGGGCGAGTCC CCTTCCACGACTTGCCC
    CGTA (SEQ ID NO: 45) AGCAT (SEQ ID NO: 46)

    PGCLC Induction with BMP-4 Transwell Differentiation
  • Corning Costar Transwell plates with a 6.5 mm diameter and 0.4 μm pore size (Cat. #07-200-147, Ref. #3414) were used. Transwell membranes were coated overnight with Matrigel. Prior to seeding, the Matrigel was removed and the membrane was rinsed 3× with PBS+/+ and then put into mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM). Cells were then immediately seeded onto the transwell membranes at a density of 500-1,500 cells/mm2 (16,600-49,800 cells/well). Twenty-four hours later, ROCK inhibitor was removed, and the cells were fed with fresh mTESR1. Twenty-four hours after ROCK inhibitor removal, BMP-4 was added to both the apical (top) and basolateral (bottom) compartments. Forty-eight hours after BMP-4 induction, the transwells were fixed prior to staining for PGCLC and other somatic lineage markers (FIG. 3 ). Prior to imaging, the transwell membrane was removed and mounted onto a glass coverslip. 10 ng/mL BMP4 in transwells with a cell density of 750-1,000 cells/mm2 was optimal for PGCLC induction.
    • Immunofluorescent Imaging
  • For staining, colonies and monolayers (plate or transwell) were fixed with 4% paraformaldehyde (VWR) for 20 minutes and subsequently rinsed 3× with PBS. Fixed cells were blocked and permeabilized for one hour at room temperature in 5% normal serum and 0.3% Triton™ X-100 (Sigma Aldrich) in PBS. Samples were then incubated with primary antibodies (still in staining buffer 5% normal serum/0.3% Triton™ X-100) overnight at 4° C. The following day, cells were rinsed 3× with PBS and incubated with secondary antibodies (1:400) in a 1% BSA, 0.3% Triton™ X-100 PBS solution. Primary and secondary antibodies used are listed in Table S.
  • TABLE 5
    Antibodies for Immunofluorescent Staining
    Target Species Catalog Number Supplier
    BLIMP1 Ms MAB36081 R&D
    BMPR1A Rb 38-600 ThermoFischer
    CDX2 Rb 12306 Cell Signaling
    EOMES Ms MAB6166 LEDQ0218092
    Ezrin Ms MA5-13862 ThermoFischer
    pSMAD1/5Oct4 RbGt 41D10, 9516sSC- Cell Signaling
    8629 Santa Cruz Biotech
    SOX17pSMAD1/5 GtRb AF192441D10, R&D Cell Signaling
    9516s
    SOX2SOX17 RbGt AB59776AF1924 Abcam R&D
    SOX2SOX2 MsRb 4900AB59776 Cell Signaling Abcam
    TBXTSOX2 GtMs AF20854900 R&D Cell Signaling
    ZO-1TBXT MsGt 33-9100AF2085 Invitrogen R&D
    ZO-1 Ms 33-9100 Invitrogen
    • BMP4 Differentiation in Unconfined Colonies
  • To generate unconfined colonies of a defined size, PSCs were first force aggregated into 400×400 mm PDMS microwell inserts (24-well plate sized, ˜975 microwells/insert) using previously published protocols (Libby et al., bioRxiv 1-23 (2018); Hookway et al., Methods 101, 11-20 (2016); Ungrin et al., PLOS One 3, (2008)). Briefly, PSCs were dissociated, resuspended in mTESR1 supplemented with ROCK inhibitor (10 uM), seeded into the microwell inserts at a concentration of ˜50-100 cells/well, centrifuged at 200 relative centrifugal field (rcf) for 3 minutes, and left overnight to condense into aggregates. Next, the aggregates (˜50-100 cells in size) were resuspended in mTESR1 supplemented with ROCK inhibitor (10 uM) and transferred to Matrigel-coated 96 well plates at a concentration of approximately ˜15 aggregates/well, where they were allowed to attach and form 2D colonies. After 24 hours, ROCK inhibitor was removed and the colonies were fed with mTESR1. mTESR1 supplemented with BMP4 (200 ul/well, 50 ng/ml, R&D Systems) was added another 24 hours later to start the differentiation. Unconfined colonies of a defined size were also generated using an alternative protocol. Briefly, dissociated hPSCs were seeded at 2 cells/mm2, and fed with mTESR1 supplemented with ROCK inhibitor for 4 days, after which they were fed for 2 days with regular mTESR1 or until they reached an appropriate size (approximately 300-500 um in diameter), after which they were treated with BMP4 as described above.
  • Transwell Culture of hPSCs and FITC Diffusion Assay
  • Corning Costar Transwell plates with a 6.5 mm diameter and 0.4 μm pore size (Cat. #07-200-147, Ref. #3414) were used. Transwell membranes were coated overnight with Matrigel. Prior to seeding, the Matrigel was removed and the membrane was rinsed 3× with PBS+/+ and then put into mTESR1 supplemented with ROCK inhibitor Y-27632 (10 uM). Cells were then immediately seeded onto the transwell membranes at a density of 1,500 cells/mm2 (49,800 cells/well). 24 hours later the ROCK inhibitor was removed, and the cells were fed with fresh mTESR1. 24 hours after ROCK inhibitor removal, the membranes were imaged on an EVOS fluorescence microscope at 10× to visualize whether the GFP labelled cellular nuclei reached confluence and were completely covering the membrane. The inventors had previously determined that this protocol generates intact epithelia at this timepoint.
  • To visualize pSMAD1 activity in BMP4 stimulated transwells over time, BMP4 (50 ng/ml) was added to either the apical (top) or basolateral (bottom) compartments of the transwell. The transwells were fixed at the appropriate time points by transferring the insert to a new 24 well plate, rinsing with PBS, and fixing with 4% PFA.
  • To perform the FITC diffusion assay, FITC conjugated to 40-kDa dextran (Sigma-Aldrich) was added to the apical compartment and 10 ul of media was collected from basolateral compartment at various timepoints, which was mixed with 90 ul of PBS onto a 96-well dark-sided plate. After the time course was completed, a plate reader was used to take fluorescence measurements of our samples over time.
    • Immunofluorescent Staining and Marker Quantification
  • Human PSCs were rinsed with PBS 1×, fixed in 4% paraformaldehyde (VWR) for 15 minutes, and subsequently washed 3× with PBS. The fixed cells were permeabilized and blocked in 0.3% Triton X-100 (Sigma Aldrich) and 5% normal donkey serum for an hour, and then incubated with primary antibodies overnight (also in 0.3% Triton, 5% normal donkey serum). The following day, samples were washed 3× with PBS and incubated with secondary antibodies in 0.3% Triton and 1% BSA at room temperature for 2 hours. Secondary antibodies used conjugated with Alexa 647, Alexa 405, and Alexa 555 (Life Technologies), and were used at a dilution of 1:400.
    • RNA Sequencing
  • ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells were seeded at a density of 250 cells/mm2 onto standard culture 6-well plates in mTESR1 supplemented with ROCK inhibitor (10 uM). 24 hours later, ROCK inhibitor was removed, and the cells were fed with fresh mTESR1. 24 hours after ROCK inhibitor removal, cell lysates for the pluripotent condition were prepared by putting 1.5 mL RLT (lysis) buffer/well for 3 minutes, and freezing this lysate at −80° C. for subsequent RNA extraction. Simultaneously, BMP4 (50 ng/ml) was added to the differentiated condition. After 48 hours of BMP4 treatment, cell lysates for the differentiated condition were prepared as described above. RNA extraction was performed using Qiagen's RNBasy kit, and samples were subsequently shipped to Novogene for library preparation and sequencing (Illumina, PE150, 20M paired reads).
    • Whole Genome Bisulfite Sequencing
  • ZO1 wild type (ZWT) and ZO1 knockdown (ZKD) cells were seeded and cultured as described in the RNA sequencing section. Only pluripotent samples were sent for sequencing. To do this, cells were dissociated using Accutase and resuspended in 200 ul PBS+proteinase K, and then frozen at −20° C. for subsequent DNA extraction. DNA extraction was performed using Qiagen's DNA extraction kit. Samples were subsequently sent to CD Genomics for whole genome bisulfite sequencing (Illumina, PE150, 250M paired reads).
    • Example 2: ZO1-Knockdown and BMP to Make PGC Like-Cells (PGCLCs)
  • This Example illustrates generation of primordial germ-like cells (PGCLCs) from hiPSC cells modified to knockdown ZO1.
  • A doxycycline (DOX)-inducible CRISPR interference system was made for integration into the WTB (female) and WTC (male) parent hiPSC lines (FIG. 1A). The CRISPR interference system was comprised of two components: a dCas9KRAB repressor driven by a TetO promoter that was inserted into the AAVS1 safe harbor locus and that is expressed only under DOX treatment, and a constitutively expressed guide RNA (gRNA) that targets the transcriptional start site of the ZO1 gene. The ZO1-specific gRNA (Table 2; FIG. 1A) was encoded in a randomly-integrating plasmid that also expressed a blasticidin selection gene. DOX-inducible expression of Cas9 enabled temporal control of its gene expression. These constructs were transfected into both the WTB and WTC hiPSC CRISPRi cell lines. Knockdown of ZO1 was achieved after 5 days of DOX treatment in cells cultured in mTESR on Matrigel coated plates (seeding density 120 cells/mm2). The cells were passaged every three days using Accutase for cell displacement. hPGCLC induction was commenced by adding 50 ng/mL BMP4 directly to a monolayer of ZO1 knockdown hiPSCs at seeding densities between (100-2000 cells/mm2) for at least two days.
  • As illustrated in FIG. 1B-1C, reduced expression of ZO1 was observed in the cells within one day of DOX treatment, and ZO1 expression became minimal by day 5 after DOX was introduced into the culture medium.
  • To evaluate the barrier function and ability of ZO1 knockdown cells to preclude diffusion of molecules from one side of a cellular monolayer to the other, an assay was performed that involved growing the wild type or ZO1 cells on a transwell membrane where both apical and basolateral sides are independently accessible. The apical side was treated with 40 kDa FITC (dextran molecules conjugated with the fluorescent molecule FITC), and media from the basolateral side was sampled over time for fluorescent measurements to determine permeability of the cell layer. FIG. 1D show that ZO1 knockdown results in loss of tight junction barrier function as measured by FITC-Dextran diffusion. Hence, apical to basolateral diffusion is disrupted by ZO1 knockdown.
  • Wild type and ZO1-knockdown cells that were maintained in transwells were treated for 5 days with Doxycycline (2 uM) and the transepithelial electrical resistance (TEER) of the cells was measured. As shown in FIG. 1E, ZO1 knockdown cells exhibit loss of transepithelial resistance, indicating ZO1 knockdown results in loss of barrier function.
  • FIG. 2A illustrates that when BMP4 is provided basolaterally (diagram inset), pSMAD1 expression is activated whether or not ZO1 expression is knocked down (see top row of images). However, when BMP4 is provided apically, pSMAD1 expression is not activated when ZO1 is expressed (FIG. 2A, bottom left panels). However, pSMAD1 expression is activated when ZO1 is not expressed (FIG. 2A, bottom right images).
  • FIG. 2B illustrates methods tested for generating PGCLCs from pluripotent stem cells. Knock down (KD) of ZO1 expression is not necessary for generating PGCLCs when BMP4 is provided basolaterally in a culture medium such as mTESR (FIG. 2B top row with BMP4 on the bottom row). However, ZO1 knockdown (KD) can be used to facilitate PGCLC generation by DOX-induced KD (FIG. 2B, middle row). Addition of BMP4, especially basolateral addition of BMP4, to ZO1 knockdown PSCs can also generate PGCLCs.
  • Moreover, the cells need not be aggregated and can just be seeded directly onto Matrigel coated plates and stimulated with BMP4 for 48 hours. FIG. 2C˜2E show successful differentiation of ZO1 KD hiPSCs to PGCLCs, using both aggregation and monolayer differentiation methods.
    • Example 3: Generating Primordial Germ Cells without Genetic Modification
  • This Example describes methods for differentiating pluripotent stem cells (PSCs) to primordial germ cells like cells (PGCLCs), where the pluripotent stem cells (PSCs) are not genetically modified, or chemically treated (except for the addition of ROCK inhibitor to promote survival after seeding).
  • One day prior to dissociating the PSCs, Matrigel was coated onto the transwell membranes, and left at 37° C. overnight. The next day, pluripotent stem cells (PSCs) growing in mTESR medium were dissociated with Accutase and resuspended in mTESR with 10 uM ROCK inhibitor. Matrigel was aspirated off of the transwell membranes and the apical and basolateral compartments were filled with mTESR+10 uM ROCK inhibitor. The PSCs were seeded at a density of 1000 cells/mm2 onto the transwell membrane, however in some cases, the number of seeded PSCs can be varied. The following day, the spent media was aspirated, and mTESR media was added. The day after that, mTESR media was added to the apical compartment, and mTESR media with 5-50 ng/mL BMP4 was added to the basolateral compartment, as shown in FIG. 3 in the rightmost panel. 10 ng/mL BMP4 was found to be optimal for PGCLC induction. PGCLCs could be harvested starting at Day 2, but the cells can be incubated with daily changes of differentiation media up until Day 6 to increase cell yield.
    • Example 4: BMP Pathway Activation Correlates with Regional Loss of ZO1
  • Human PSCs confined to circular micropatterns and treated for 42-48 hours with BMP4 undergo radial patterning of gastrulation-associated makers CDX2 (trophectoderm-like), TBXT (mesendoderm-like), and SOX2 (ectoderm-like), specified radially inward from the colony border. The inventors and others have demonstrated that similarly-sized colonies whose growth is not confined by micropatterns undergo analogous radial patterning in response to BMP4 stimulation (Libby et al., bioRxiv 1-23 (2018); Joy et al. Stem Cell Reports 16, 1317-1330 (2021); Gunne-Braden et al., Cell Stem Cell 26, 693-706.e9 (2020)) (FIG. 5A-5B). In this modified protocol, human pluripotent stem cells were aggregated overnight within pyramidal microwells, and the following day these 3D aggregates are re-plated sparsely and allowed to grow into distinct 2D colonies 300-500 um in diameter. This system was utilized because, compared with micropatterned colonies, unconfined colonies maintain a relatively uniform density and a robust epithelial morphology over time (FIG. 5E-5G). This is important because epithelial integrity is a direct function of cell density; previous reports have linked changes in signaling and cell specification with changes in cell density (Etoc et al., Dev. Cell 39, 302-315 (2016); Nallet-Staub et al., Dev. Cell 32, 640-651 (2015); Smith et al., Proc. Natl. Acad. Sci. U.S.A. 115, 8167-8172 (2018); Manfrin et al., Nat. Methods 16, 640-648 (2019)).
  • Low cell densities can prevent proper tight junction formation and presumably enhance permeability to signaling proteins (Etoc et al., Dev. Cell 39, 302-315 (2016). Interestingly, the inventors have discovered that the opposite is also true: in monolayer culture at high cell densities, the honeycomb-like intercellular protein expression pattern of ZO1, which is indicative of an intact epithelium, becomes disrupted and punctate (FIG. 5H). Regions with punctate ZO1 expression, which increase in frequency as cell density increases, overlap with regions of BMP4-induced signaling pathway activation (phosphorylation of SMAD1). This suggests that very low and very high cell densities can both cause increases in epithelial permeability. In our hands, this phenotype is also present in micropatterned colonies, regions of high density lose ZO1 and overlap with pSMAD1 activation upon BMP4 stimulation (FIG. 5F). Discrepancies in previously reported pSMAD1 pre-patterns may therefore be explained in part to regional changes in density and consequent effects on epithelial structure.
  • Interestingly, ZO1 expression inversely correlates with pSMAD1 activation even in the context of unconfined colonies with uniform density. For example, at early timepoints upon induction with BMP4, pSMAD1 activity is largely limited to the edge of colonies. ZO1 expression does not fully extend to the edge of the colony, and tapers off a distance of approximately one cell layer before reaching the edge.
  • Co-staining of ZO1 and pSMAD1 in unconfined colonies after 1 hour of BMP4 stimulation exhibited an anti-correlation between pSMAD1 positive and ZO1 positive regions (FIG. 5C)—cells expressing pSMAD1 did not also express ZO1. Quantification of fluorescent signal normalized to nuclear LMNB1 expression at different distances from the colony edge further demonstrated the inverse relationship between pSMAD1 and ZO1 (FIG. 5D). Initial pSMAD1 pre-patterning has been implicated in regulating subsequent gastrulation-associated patterning in micropatterned colonies. The inventors have conducted the experiments described herein to elucidate the effect of tight junctions on signaling and gastrulation patterning.
    • Example 5: ZO1 Knockdown Leads to Ubiquitous and Sustained Pathway Activation
  • In vitro hPSCs cultured as epithelial sheets that have tight junctions and display apical/basolateral polarity, with most morphogen receptors, including BMP receptors BMPR1A, BMPR2, and ACVR2A, localized to the basolateral side. These receptors are physically partitioned away from morphogens presented in the media on the apical side. As a result, tight junction expression presumably attenuates cellular response to exogenous morphogen signals in vitro (FIG. 6A).
  • In order to explore how tight junctions affect signaling in the unconfined colonies, the DOX inducible CRISPR interference (CRISPRi) system was used to knockdown ZO1 (FIG. 1A). ZO1 was specifically targeted because preliminary RNA sequencing data showed that ZO1 is much more highly expressed in cultured hPSCs than ZO2 or ZO3 (data not shown). Both male (WTC) and female (WTB) hPSC ZO1 knockdown lines were created. The WTC line also contained a LMNB1-GFP fusion reporter for live nuclear visualization. Both hPSC ZO1 CRISPRi lines were karyotypically normal (FIGS. 1G-1H), and RNA and protein expression are significantly depleted after five days of DOX treatment, as shown by qPCR, immunofluorescence (IF), and western blot (FIGS. 1B-1C, 6B). Most of the characterization in the WTC ZO1 CRISPRi line was performed with and without DOX (referred to in the text as ZO1 wild type (ZWT) and ZO1 knockdown (ZKD), respectively), however, the results for the WTB ZO1 CRISPRi line were phenotypically similar and reproducible.
  • ZO1 knockdown cells grew in somewhat denser colonies and exhibited rounder nuclear shapes (FIG. 6C-6D). Where ZO1 wild type nuclei are stretched and flat, ZO1 knockdown nuclei are taller and more rounded, likely as a result of severed connections between the cell-cell junctions and the actin cytoskeleton/nuclear lamina.
  • When grown as unconfined colonies and exposed to BMP4, ZO1 wild type largely limited pSMAD1 expression to the colony edge at early timepoints (15 min-1 hr) (FIG. 5C-SD). At later timepoints (6 hours), pSMAD1 is detectable in cells located centrally within the colony. However, pSMAD1 expression is subject to inhibitor feedback loops. Thus, this pathway activation is shut off by 48 hours in ZO1 wild type cells (FIG. 6E-6F). Strikingly, at early timepoints, the ZO1 knockdown colonies displayed pSMAD1 throughout the colony (FIG. 6F). Furthermore, ZO1 knockdown cells maintain pSMAD1 activation over time (FIG. 6F), despite significant increases in transcription of the secreted BMP inhibitor NOGGIN (FIG. 7J), which is implicated in driving SMAD1 pathway inactivation in ZO1 wild type cells over time. In ZO1 wild type cells, NOGGIN is secreted apically and is trafficked transepithelially with assistance from glycoproteins on the apical surface.
  • The observed maintenance of pSMAD1 pathway activation despite increase in NOGGIN in ZO1 knockdown colonies indicates that ZO1 is not only important for preventing ligands such as BMP4 from accessing basolateral receptors, but may also be necessary in rendering the cells sensitive to some inhibitors, presumably by maintaining expression of the apical surface glycoproteins that enable transepithelial trafficking of apically secreted inhibitors such as NOGGIN or sequestration/concentration of other basolaterally secreted morphogen inhibitors within the colony interior. This observation is reinforced by the fact that ZO1 knockdown cells also exhibit loss of apical Ezrin expression (FIG. 1F), which can be important in tethering apical glycoproteins to the actin cytoskeleton.
    • Example 6: Signaling Changes Result from Increased Permeability in ZO1 Knockdown Cells
  • In order to confirm basolateral sequestration of BMP receptors within an epithelium, cells were grown on a transwell membrane, where both apical and basolateral sides of the media are accessible. Using transwells allows for unidirectional exposure of BMP4 from either cellular domain. As early experiments have indicated, basolateral presentation of BMP4 is required for pSMAD1 activation in ZO1 wild type cultures. Alternatively, both apical and basolateral stimulation activates pSMAD1 in ZO1 knockdown (ZKD) cells (FIG. 7H). ZO1 wild type and ZO1 knockdown cells do not have differences in BMP4 receptor expression (FIG. 7I). Several possibilities could explain this phenomenon: ZO1 knockdown causes mixing of apical/basolateral domain elements through the plasma membrane and disrupted trafficking of receptors to their proper domains (loss of apical/basolateral polarity), or ZO1 knockdown causes increased permeability to signaling molecules (loss of barrier function). To test these possibilities, the inventors first characterized apical/basolateral polarity between ZO1 wild type and ZO1 knockdown cells.
  • In polarized cells, the Golgi apparatus faces the apical (secretory domain) direction. Therefore, the inventors evaluated positioning of the Golgi in ZO1 wild type and ZO1 knockdown cells. Z-stacks revealed that in both cell types, the Golgi sits on top of the nucleus on the apical side of the cell, suggesting that polarity of the ZO1 knockdown cells is still intact (FIG. 7K-7L). However, staining for the apical marker Ezrin revealed significant eradication of the apical domain in ZO1 knockdown cells, characterized by punctate Ezrin localization. This is consistent with previous reports that Ezrin is lost on the colony edge of regular hPSC colonies (Kim et al., Stem Cell Reports 17, 68-81 (2022)). Immunofluorescence images showed that swaths of ZO1 knockdown cells lost apical Ezrin; and even in regions where Ezrin is present, it overlaps significantly with BMPR1A (a basolateral BMP receptor), indicating potential changes in localization of some apical/basolateral elements (FIG. 7M-7N). Our results indicate that polarity-associated changes do not occur in cytoplasmic elements, but may occur for elements bound to the plasma membrane.
  • FITC based diffusion assay was performed to look for differences in permeability in ZO1 wild type and ZO1 knockdown. Each cell type was grown on a transwell membrane and a 40 kDa dextran conjugated with FITC was added to the apical compartment (FIG. 6G). The 40 kDa-FITC was selected due to its similarity in hydraulic radius to many common gastrulation-associated signaling proteins. Specifically, 40 kDa-FITC is slightly smaller than BMP4. Hence, an epithelial barrier that could exclude the 40 kDa-FITC is evidence that the epithelial barrier could also exclude BMP4.
  • Fluorescence measurements of the basolateral compartment over time were used to quantify permeability of the ZO1 knockdown cells compared to the control. As shown in FIG. 6F, significant increases in passage of FITC through ZO1 knockdown cell layers could be observed as early as 30 minutes into 40 kDa-FITC treatment. Similarly, trans epithelial resistance (TEER) measurements performed on control and ZO1 knockdown monolayers confirmed that ZO1 knockdown cells are not able to form a true epithelium that resists passage of ions through the paracellular space (FIG. 6I). Therefore, while some changes in apical/basolateral polarity may occur, the results described herein indicate that definitive changes in permeability drive heightened signaling pathway activation seen in ZO1 knockdown cells.
    • Example 6: ZO1 Knockdown Causes Changes in Cell Fate Proportions in Unconfined Gastrulation Models
  • Several models have been proposed to explain how multiple distinct lineages can arise in a colony exposed to a uniform dose of BMP4. The current paradigm combines the principles of Alan Turing's reaction diffusion (RD) (Turing, Philos. Trans. R. Soc. 37-72 (1952)) and Lewis Wolpert's positional information (PI) (Wolpert, J. Theor. Biol. 25, 1-47 (1969); Green & Sharpe, Dev. 142, 1203-1211 (2015)). The RD model proposes that in response to signal pathway activation (phosphorylation of SMAD1) by an activating species (BMP4), cells secrete more of this activator (BMP4) and its inhibitor (NOGGIN) in a feedback loop (Tewary et al., Development dev. 149658 (2017)). Differences in the diffusivities between NOGGIN and BMP4 can create a steady-state gradient of effective BMP4 concentrations across the colony, and cells sense positional information and differentiate based on both on this concentration gradient and its overlap with other members of a BMP4-induced feedback loop, including WNT and NODAL. The initial pSMAD1 pre-pattern is therefore assumed to be an important indication of the shape of an RD gradient which determines the shape of subsequent gastrulation-associated patterning.
  • In ZO1 wild type, this temporal pSMAD1 profile is reserved for cells on the edge of colonies that remain pSMAD1 positive throughout BMP4 stimulation and eventually acquire CDX2+ trophectoderm-like fates. By contrast, ZO1 knockdown cells maintain ubiquitous and sustained pSMAD1 activation throughout the entire colony. Therefore, if the current RD/PI paradigm is correct, the inventors predicted that ZO1 knockdown cells would ubiquitously differentiate to the CDX2 lineage (FIG. 7A). Accordingly, these results show that ZO1 knockdown colonies treated with BMP4 have increased CDX2 expression across the colony interior. In addition, these ZO1 knockdown colonies display a stark decrease in central SOX2 expression, and disruption of the TBXT ring pattern (FIG. 7B-7C). These results establish ZO1, and therefore tight junction stability, as a key component of BMP4-induced cell fate and spatial patterning.
    • Example 7: RNA Sequencing of BMP4-Treated ZO1 Knockdown Colonies Reveals PGCLC Bias
  • Unexpectedly, the inventors also observed that like CDX2, TBXT expression is substantially increased throughout the center of the colony (FIG. 7B). Many progenitor cell types express TBXT. To better identify this TBXT-expressing population and quantify changes in ZO1 knockdown induced lineage bias, RNA sequencing was performed on pluripotent and 48-hour BMP4 treated ZO1 wild type and ZO1 knockdown cells.
  • RNA sequencing confirmed the immunofluorescence staining results: CDX2 and TBXT transcripts are upregulated, whereas SOX2 is downregulated (FIG. 7D). Analysis of a panel of well-known gastrulation associated lineage markers in ZO1 wild type and ZO1 knockdown cells revealed that ZO1 knockdown cells have the tendency to express mesendoderm, PGC, and extraembryonic markers at the expense of ectodermal-like lineages (FIG. 7E).
  • Gene ontology (GO) analysis performed on Clusters 2 and 3 of the top 150 differentially expressed genes between ZO1 wild type and ZO1 knockdown cells shows upregulation of endoderm and sex cell related pathways in ZO1 knockdown colonies, as illustrated in Table 6 below.
  • TABLE 6
    Gene Sets Enriched in ZO1 Knockdown Cells
    Gene-set Enriched GO Terms FDR
    Cluster 2:
    Endodermal cell differentiation 4.62E−02
    Mesoderm formation 1.49E−04
    Embryonic placenta development 2.23E−02
    Cell migration involved in gastrulation 1.75E−04
    Trophectodermal cell differentiation 1.41E−02
    Cluster 3:
    Endodermal cell fate determination 7.99E−03
    Embryonic foregut morphogenesis 1.60E−03
    Reproductive system development 5.79E−03
    Sex differentiation 1.95E−03
    Germ cell migration 3.07E−02

    Similarly, unbiased clustering of the top 16 differentially expressed genes between ZO1 wild type and ZO1 knockdown revealed significant increases in NANOS3, SOX17, and WNT3 (FIG. 7F), genes that when expressed together are associated with the human PGC specification program (Irie et al., Cell 160, 253-268 (2015)). Subsequent immunofluorescence staining for PGC markers BLIMP1, TFAP2C, and SOX17 at 48 hours showed increased expression of these markers in ZO1 knockdown colonies at 48 hours compared with the ZO1 wild type controls (FIG. 7G). This phenotype can also be observed outside of the colony format at 48 hours. By 72 hours, clear triple positive expression of BLIMP1/TFAP2C/SOX17 can be seen in the majority of ZO1 knockdown cells (FIG. 8A-8B) in monolayer culture, a phenotype that is also observed in the WTB ZO1 knockdown hPSC line (FIG. 8E-8F). Together, these results indicate that disrupting tight junction “stability” in the presence of BMP4 dramatically augments cell receptiveness to signals needed for PGCLC emergence.
    • Example 8: Decoupling Signaling and Structural Changes in ZKD PGCLCs
  • Upon the discovery of a nascent PGCLC population within the ZO1 knockdown colonies, the inventors sought to decouple the effects of structural changes due to tight junction instability and ubiquitous pSMAD1 activation in enabling this PGCLC population to emerge. Two papers describe different protocols for generating human PGCLCs (Irie et al., Cell 160, 253-268 (2015); Sasaki et al. Cell Stem Cell 17, 178-194 (2015)). In the first protocol by Sasaki et al., hPSCs were pre-induced into an incipient mesoderm-like (iMeLC) state that renders the cells poised for PGCLC specification. In the second protocol by Irie et al., hPSCs are first reset from a primed to a naïve pluripotency state, as primed hPSCs are thought to have lost the developmental potential to generate PGCLCs. Without iMeLC or naïve pluripotency pre-induction, both protocols failed to efficiently generate PGCLCs, providing only about 1-2% efficiency of generating PGCLCs.
  • However, using the differentiation methods described herein, ZO1 knockdown cells do not undergo any form of pre-induction yet are able to produce a robust PGCLC population.
  • Two possibilities potentially explain this PGCLC specification bias: 1) ZO1 knockdown is causing a change in pluripotent ground state (to a naïve-like or iMeLC-like state), or 2) signaling changes caused by ZO1 knockdown recapitulate in vivo PGC specification, and are sufficient to drive PGCLC differentiation in vitro.
  • The inventors first characterized pluripotency in ZO1 wild type and ZO1 knockdown cells in the absence of BMP4. RNA sequencing showed that aside from ZO1 and ZNF10 (which is part of the CRISPRi machinery), few genes are both significantly and substantially differentially expressed between ZO1 wild type and ZO1 knockdown cells (FIG. 8G), and no significant changes are shown in major canonical pluripotency markers (FIG. 8C). Whole genome bisulfite sequencing shows that while several probes are differentially methylated (FIG. 8D, 8H), there are no global changes in methylation of probes between ZO1 wild type and ZO1 knockdown cells, which would be expected if a resetting process occurred. GO analysis also did not reveal any significant links between genes with methylated probes. Together, these data indicate that the transcriptome and methylome are not greatly affected and there is no observable change in ground state that explains ZO1 knockdown predisposition to PGCLC lineages.
  • Next the inventors tested the hypothesis that ZO1 knockdown cells are predisposed to PGCLC fates because, unlike ZO1 wild type cells which undergo NOGGIN-related BMP4-pathway inhibition at later timepoints, ZO1 knockdown cells are able to maintain BMP4-pathway activation.
  • To decouple changes in signaling from potential structural changes that result from ZO1 knockdown, the inventors designed experiments to recapitulate the pSMAD1 signaling dynamics in hPSCs without ZO1 knockdown. ZO1 wild type cells were grown on a transwell membrane where both the apical and basolateral sides were exposed to the media. As described, bi-directional stimulation of hPSCs with BMP4 resulted in ubiquitous and sustained activation of pSMAD1 over the course of 48 hours, much like when ZO1 knockdown cells are stimulated in standard culture (FIG. 9A). RNA sequencing of stimulated ZO1 wild type and ZO1 knockdown cells grown on transwells showed remarkable similarities in marker expression between the two samples, demonstrating that most of the observed changes in cell fate are a direct result of increased signal pathway activation. The total number of differentially expressed genes between ZO1 wild type and ZO1 knockdown samples was significantly higher in standard culture (3150) versus in transwell (35) culture, highlighting the magnitude of the expression changes dependent solely on changes in pSMAD1 signaling. Of these 35 genes, unbiased clustering and GO analysis demonstrated that ZO1 knockdown cells still have a slight bias towards mesendodermal lineages, as illustrated in Table 7 below.
  • TABLE 7
    Gene Sets Enriched in ZO1 Knockdown Cells
    Gene-set Enriched GO Terms FDR
    Cluster 2:
    Primitive streak formation 4.62E−02
    Cluster 3:
    Embryonic foregut morphogenesis 7.50E−04
    Cellular response to erythropoietin 2.93E−02
  • Interestingly, neither ZO1 wild type nor ZO1 knockdown cells grown on transwell membranes and treated for 48 hours with BMP4 (50 ng/ml) were as predisposed to PGCLC fates as was seen for ZO1 knockdown cells on standard plates. The hypothesized that this was a result of too much signal from bi-directional stimulation on the transwell. Decreasing the BMP4 concentration to 10 ng/mL resulted in robust and ubiquitous PGCLC differentiation of ZO1 wild type cells on the transwell membranes (FIG. 9B). Taken together, these results indicate that changes in cell identity in the absence of ZO1, and specifically the emergence of a PGCLC population, are largely due to increased susceptibility to BMP4 signaling.
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  • All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.
  • The following statements are intended to describe and summarize various embodiments of the invention according to the foregoing description in the specification.
    • Statements:
  • 1. A system comprising pluripotent stem cells supported on a porous surface in a culture medium that contains BMP.
  • 2. The system of statement 1, wherein the pluripotent stem cells are human pluripotent stem cells.
  • 3. The system of statement 1 or 2, wherein the pluripotent stem cells are induced pluripotent stem cells.
  • 4. The system of statement 1, 2 or 3, wherein the pluripotent stem cells are genetically modified.
  • 5. The system of any one of statements 1-4, wherein the pluripotent stem cells are genetically modified to correct a genetic defect.
  • 6. The system of any one of statements 1-5, wherein the pluripotent stem cells are genetically modified to reduce the expression or function of an endogenous tight junction gene.
  • 7. The system of statement 6, wherein the tight junction gene is at least one endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • 8. The system of any one of statements 1-7, wherein the porous surface has pores that the cells cannot pass through.
  • 9. The system of any one of statements 1-8, wherein the porous surface has pores of about 0.4 μm to about 8.0 μm in diameter.
  • 10. The system of any one of statements 1-9, wherein the porous surface is a membrane.
  • 11. The system of any one of statements 1-10, wherein the porous surface is an insert of a transwell plate.
  • 12. The system of any one of statements 1-11, wherein the system comprises a transwell plate.
  • 13. The system of any one of statements 1-12, wherein the BMP is BMP2, BMP4, or a combination thereof.
  • 14. The system of any one of statements 1-13, which comprises an apical compartment and a basolateral compartment.
  • 15. The system of any one of statements 1-14 wherein the pluripotent stem cells are within or receive BMP from a basolateral compartment.
  • 16. The system of any one of statements 1-15, wherein the BMP is at a concentration of at least 0.1 ng/ml, or at least 1 ng/ml, or at about 2 ng/ml or at least 5 ng/ml, or at least 10 ng/ml, or at least 20 ng/ml, or at least 25 ng/ml, or at least 30 ng/ml, or at least 35 ng/ml, or at least 40 ng/ml, or at least 50 ng/ml.
  • 17. The system of any one of statements 1-16, wherein the BMP is at a concentration of less than 200 ng/ml, or less than 150 ng/ml, or less than 100 ng/ml, or less than 75 ng/ml, or less than 60 ng/ml.
  • 18. The system of any one of statements 1-17, wherein the porous surface is conditioned with extracellular matrix protein prior to seeding the pluripotent stem cells on the porous surface.
  • 19. The system of statement 18, wherein the extracellular matrix protein is removed from the porous surface prior to seeding the pluripotent stem cells on the porous surface.
  • 20. The system of any one of statements 1-19, wherein the pluripotent stem cells are incubated with a ROCK inhibitor prior to seeding the pluripotent stem cells on the porous surface.
  • 21. The system of any one of statements 1-20, further comprising at least one primordial germ cell.
  • 22. The system of any one of statements 1-21, further comprising a population of primordial germ cells.
  • 23. A method comprising inhibiting or bypassing tight junction formation in a population of pluripotent stem cells to generate a modified cell population, and contacting the tight-junction modified cell population with BMP.
  • 24. The method of statement 23, wherein inhibiting or bypassing tight junction formation comprises:
      • a. incubating the population of pluripotent stem cells on a porous surface to bypass apical tight junctions;
      • b. contacting the population of pluripotent stem cells with one or more inhibitory nucleic acids that bind one or more tight junction nucleic acids (one or more tight junction mRNA or DNA);
      • c. contacting the population of pluripotent stem cells with one or more CRISPRi ribonucleoprotein (RNP) complexes targeted to one or more tight junction gene;
      • d. contacting the population of pluripotent stem cells with one or more expression vectors or virus-like particles (VLP) encoding one or more guide RNAs that can bind one or more tight junction gene; and
      • e. combinations thereof.
  • 25. The method of statement 24, wherein the porous surface has pores that the cells cannot pass through.
  • 26. The method of statement 24 or 25, wherein the porous surface has pores of about 0.4 μm to about 8.0 μm in diameter.
  • 27. The method of statement 24, 25 or 26, wherein the porous surface is a membrane.
  • 28. The method of any one of statements 24-27, wherein the porous surface is an insert of a transwell plate.
  • 29. The method of any one of statements 28, wherein the transwell plate comprises an apical compartment and a basolateral compartment.
  • 30. The method of statement 29, wherein the basolateral compartment comprises culture medium comprising BMP.
  • 31. The method of any one of statements 24-30, wherein the porous surface is conditioned with extracellular matrix protein prior to seeding the pluripotent stem cells on the porous surface.
  • 32. The method of statement 31, wherein the extracellular matrix protein is removed from the porous surface prior to seeding the pluripotent stem cells on the porous surface.
  • 33. The method of any one of statements 24-32, wherein the inhibitory nucleic acids that bind one or more tight junction nucleic acids comprise one or more short interfering RNA (siRNA), IRNA, antisense nucleic acid, or a combination thereof.
  • 34. The method of any one of statements 24-33, wherein the population of pluripotent stem cells contacted with one or more CRISPRi ribonucleoprotein (RNP) complexes comprises pluripotent stem cells that express a cas nuclease.
  • 35. The method of any one of statements 23-34, wherein inhibiting the tight junction formation comprises incubating the population of pluripotent stem cells with a chelator or chemical inhibitor.
  • 36. The method of statement 35, wherein the chelator or chemical inhibitor is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), dimercaptosuccinic acid, dimercaprol, genistein, 1-tert-Butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2), glycyrrhizin, or a combination thereof.
  • 37. The method of any one of statements 23-36, wherein inhibiting the tight junction formation comprises incubating the population of pluripotent stem cells with PTPN1, acetylaldehyde, genistein, protein phosphatase 2 (PP2), Clostridium perfringens enterotoxins (and their derived mutants), monoclonal antibodies against Claudin-1 (75A, OM-7D3-B3, 3A2, 6F6), monoclonal antibodies against Claudin-6 (IMAB027), Claudin-2 (1A2), monoclonal antibodies against Claudin-5 (R9, R2, 2B12), monoclonal antibodies against Occludin (1-3, 67-2), and combinations thereof.
  • 38. The method of any one of statements 23-37, wherein inhibiting the tight junction formation comprises inhibiting expression or function of at least one endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • 39. The method of any one of statements 23-38, wherein inhibiting the tight junction formation comprises inhibiting expression or function of at least one endogenous zonula occludens-1 (ZO1) allele.
  • 40. The method of any one of statements 23-39, wherein the population of pluripotent stem cells and/or the tight-junction modified cell population are incubated in a culture medium comprising a ROCK inhibitor.
  • 41. The method of any one of statements 23-40, wherein the pluripotent stem cells are human pluripotent stem cells.
  • 42. The method of any one of statements 23-41, wherein the pluripotent stem cells are autologous or allogenic to a selected subject.
  • 43. The method of statement 42, wherein the selected subject is a bird or mammal.
  • 44. The method of statement 42 or 43 wherein the selected subject is a domesticated animal, a zoo animal, an endangered animal (e.g., an animal on an endangered species list), or a human.
  • 45. The method of any one of statements 23-44, wherein the pluripotent stem cells are induced pluripotent stem cells.
  • 46. The method of any one of statements 23-45, wherein the pluripotent stem cells are genetically modified.
  • 47. The method of any one of statements 23-46, wherein the pluripotent stem cells are genetically modified to correct a genetic defect.
  • 48. The method of any one of statements 23-47, wherein the pluripotent stem cells are genetically modified to reduce the expression or function of an endogenous tight junction gene.
  • 49. The method of any one of statements 23-48, wherein the BMP is BMP2, BMP4, or a combination thereof.
  • 50. The method of any one of statements 23-49, wherein the BMP is at a concentration of at least 0.1 ng/ml, or at least 1 ng/ml, or at about 2 ng/ml or at least 5 ng/ml, or at least 10 ng/ml, or at least 20 ng/ml, or at least 25 ng/ml, or at least 30 ng/ml, or at least 35 ng/ml, or at least 40 ng/ml, or at least 50 ng/ml.
  • 51. The method of any one of statements 23-50, wherein the BMP is at a concentration of less than 200 ng/ml, or less than 150 ng/ml, or less than 100 ng/ml, or less than 75 ng/ml, or less than 60 ng/ml.
  • 52. The method of any one of statements 23-51, further comprising harvesting at least one primordial germ cell from the culture medium containing BMP.
  • 53. The method of any one of statements 28-52, further comprising differentiating at least one primordial germ cell into one or more mature germ cells.
  • 54. The method of any one of statements 28-52, further comprising administering or implanting at least one primordial germ cell into a selected subject.
  • 55. A modified pluripotent stem cell comprising a knockdown or knockout of an endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • 56. A population of modified pluripotent stem cells, each primordial germ cell comprising a knockdown or knockout of an endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
  • The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
  • The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.
  • As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a nucleic acid” or “a protein” or “a cell” includes a plurality of such nucleic acids, proteins, or cells (for example, a solution or dried preparation of nucleic acids or expression cassettes, a solution of proteins, or a population of cells), and so forth. In this document, the term “or” is used to refer to a nonexclusive or, such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.
  • Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
  • The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.
  • The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims (40)

1. A method comprising inhibiting or bypassing tight junction formation in a population of pluripotent stem cells to generate a modified cell population, and contacting the modified cell population with BMP.
2. The method of claim 1, wherein inhibiting or bypassing tight junction formation comprises:
a. incubating the population of pluripotent stem cells on a porous surface to bypass apical tight junctions;
b. contacting the population of pluripotent stem cells with one or more inhibitory nucleic acids that bind one or more tight junction nucleic acids;
c. contacting the population of pluripotent stem cells with one or more CRISPRi ribonucleoprotein (RNP) complexes targeted to one or more tight junction gene;
d. contacting the population of pluripotent stem cells with one or more expression vectors or virus-like particles (VLP) encoding one or more guide RNAs that can bind one or more tight junction gene;
e. incubating the population of pluripotent stem cells with a chelator or inhibitor; and
f. combinations thereof.
3. The method of claim 2, wherein the porous surface is a membrane or an insert of a transwell plate.
4. (canceled)
5. (canceled)
6. (canceled)
7. The method of claim 2, wherein the inhibitory nucleic acids that bind one or more tight junction nucleic acids comprise one or more short interfering RNA (siRNA), iRNA, antisense nucleic acid, or a combination thereof.
8. The method of claim 2, wherein the population of pluripotent stem cells contacted with one or more CRISPRi ribonucleoprotein (RNP) complexes comprises pluripotent stem cells that express a cas nuclease.
9. The method of claim 2, wherein the chelator or inhibitor is ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), dimercaptosuccinic acid, dimercaprol, genistein, 1-tert-Butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2), glycyrrhizin, or a combination thereof.
10. The method of claim 1, wherein inhibiting or bypassing the tight junction formation comprises inhibiting expression or function of at least one endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
11. (canceled)
12. The method of claim 1, wherein the population of pluripotent stem cells and/or the modified cell population are incubated in a culture medium comprising a ROCK inhibitor.
13. (canceled)
14. (canceled)
15. (canceled)
16. The method of claim 1, wherein the pluripotent stem cells are genetically modified.
17. (canceled)
18. The method of claim 1, wherein the pluripotent stem cells are genetically modified to reduce the expression or function of an endogenous tight junction gene.
19. The method of claim 1, wherein the BMP is BMP2, BMP4, or a combination thereof.
20. (canceled)
21. (canceled)
22. The method of claim 1, further comprising harvesting at least one primordial germ cell from the culture medium containing BMP.
23. The method of claim 22, further comprising differentiating at least one primordial germ cell into one or more mature germ cells.
24. (canceled)
25. The method of claim 22, further comprising administering or implanting at least one primordial germ cell into a selected subject.
26. A system comprising pluripotent stem cells supported on a porous surface in a culture medium that contains BMP, wherein the porous surface has pores that the cells cannot pass through.
27. The system of claim 26, wherein the porous surface is a membrane.
28. (canceled)
29. The system of claim 26, wherein the pluripotent stem cells are genetically modified.
30. (canceled)
31. The system of claim 26, which reduces expression or function of at least one tight junction gene.
32. (canceled)
33. The system of claim 26, wherein the BMP is BMP2, BMP4, or a combination thereof.
34. (canceled)
35. (canceled)
36. The system of claim 26, further comprising at least one primordial germ cell.
37. (canceled)
38. A modified pluripotent stem cell comprising knockdown or knockout of an endogenous zonula occludens-1 (ZO1), zonula occludens-2 (ZO2), zonula occludens-3 (ZO3), OCLN, CLDN2, CLDN5, CLDN6, or CLDN7 gene.
39. (canceled)
40. (canceled)
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